Antibody-based arrays for detecting multiple signal transducers in rare circulating cells

ABSTRACT

The present invention provides antibody-based arrays for detecting the activation state and/or total amount of a plurality of signal transduction molecules in rare circulating cells and methods of use thereof for facilitating cancer prognosis and diagnosis and the design of personalized, targeted therapies.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application of PCT/US07/79002,filed Sep. 20, 2007, which application claims the benefit of priority ofU.S. application Ser. No. 11/525,598, filed Sep. 21, 2006, which hasbeen converted to U.S. Provisional Application No. ______, and U.S.Provisional Application No. 60/913,087, filed Apr. 20, 2007, thedisclosures of which are hereby incorporated by reference in theirentirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

Not applicable.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

Not applicable.

BACKGROUND OF THE INVENTION

Tumor cells are often found in the blood of patients with various earlystages of cancer as “micrometastases” (disseminated tumor cells), andare also found in metastatic cancers. The number of tumor cells in blooddepends on the stage and type of the tumor. Tumors are extremelyheterogeneous. As a result, a biopsy from a single site might notrepresent the heterogeneity in a tumor population. Biopsies aretypically obtained on primary tumors; however, most metastatic tumorsare not biopsied, making molecular analysis of tumor samples even moredifficult.

During tumor metastasis, the most aggressive tumor cells leave theprimary tumor and travel through the blood and lymphatic system to reacha distant location. Thus, circulating tumor cells from blood representthe most aggressive and homogenous population of tumor cells. The numberof metastatic tumor cells in blood can vary from one to several thousandcells per milliliter of blood. Accordingly, specific and sensitivemethods are needed to detect these cells for diagnostic and prognosticpurposes. The present invention satisfies this need and provides relatedadvantages as well.

BRIEF SUMMARY OF THE INVENTION

The present invention provides antibody-based arrays for detecting theactivation state and/or total amount of a plurality of signaltransduction molecules in rare circulating cells and methods of usethereof, which have the advantages of specificity associated withenzyme-linked immunosorbent assays, sensitivity associated with signalamplification, and high-throughput multiplexing associated withmicroarrays.

In one aspect, the present invention provides an array having superiordynamic range comprising a plurality of dilution series of captureantibodies specific for one or more analytes in a cellular extract,wherein the capture antibodies are restrained on a solid support.

In another aspect, the present invention provides a method forperforming a multiplex, high-throughput immunoassay having superiordynamic range, the method comprising:

-   -   (a) incubating a cellular extract with a plurality of dilution        series of capture antibodies specific for one or more analytes        in the cellular extract to form a plurality of captured        analytes, wherein the capture antibodies are restrained on a        solid support;    -   (b) incubating the plurality of captured analytes with detection        antibodies specific for the corresponding analytes to form a        plurality of detectable captured analytes;    -   (c) incubating the plurality of detectable captured analytes        with first and second members of a signal amplification pair to        generate an amplified signal; and    -   (d) detecting an amplified signal generated from the first and        second members of the signal amplification pair.

In yet another aspect, the present invention provides a method forperforming a multiplex, high-throughput immunoassay having superiordynamic range, the method comprising:

-   -   (a) incubating a cellular extract with a plurality of dilution        series of capture antibodies specific for one or more analytes        in the cellular extract to form a plurality of captured        analytes, wherein the capture antibodies are restrained on a        solid support;    -   (b) incubating the plurality of captured analytes with detection        antibodies specific for the corresponding analytes to form a        plurality of detectable captured analytes, wherein the detection        antibodies comprise:        -   (1) a plurality of activation state-independent antibodies            labeled with a facilitating moiety, and        -   (2) a plurality of activation state-dependent antibodies            labeled with a first member of a signal amplification pair,        -   wherein the facilitating moiety generates an oxidizing agent            which channels to and reacts with the first member of the            signal amplification pair;    -   (c) incubating the plurality of detectable captured analytes        with a second member of the signal amplification pair to        generate an amplified signal; and    -   (d) detecting the amplified signal generated from the first and        second members of the signal amplification pair.

The present invention also provides kits for performing theantibody-based array methods described above comprising: (a) a dilutionseries of a plurality of capture antibodies restrained on a solidsupport; and (b) a plurality of detection antibodies. The kits canoptionally further comprise other reagents such as, for example, thefirst and second members of the signal amplification pair.

Other objects, features, and advantages of the present invention will beapparent to one of skill in the art from the following detaileddescription and figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows three antibodies specifically bound to an activatedanalyte.

FIG. 2 shows an assay scheme where labeled antibodies that havespecifically bound to an activated analyte are restrained on a solidsupport.

FIG. 3 shows the detection of total EGFR in A431 cells using monoclonalantibodies against the extracellular domain of EGFR as the captureantibody and detection antibody in an ELISA. (a) Standard curve of thesandwich ELISA. The sensitivity was about 0.25 pg/well using arecombinant extracellular domain of human EGFR. (b) Cell titrationcurve. The EGFR concentration in A431 cells was detected by ELISA. (c)Table of EGFR concentration calculated in pg/well and pg/cell. Thecalculated EGFR concentration was about 0.6 pg in each A431 cell.

FIG. 4 shows the detection of phosphorylated EGFR in A431 cells using amonoclonal antibody against the extracellular domain of EGFR as thecapture antibody and a biotin-labeled monoclonal antibody againstphosphorylated EGFR as the detection antibody in an ELISA. (a) Celltitration curve with a dilution series of the capture antibody. (b)Table of signal/noise (S/N) ratio between cells and background. Thesignal/noise ratio was 1.78 at the one cell level when the captureantibody concentration was 0.0625 μg/ml.

FIG. 5 shows the detection of total ErbB2 in SKBr3 cells usingmonoclonal antibodies against the extracellular domain of ErBb2 as thecapture antibody and detection antibody in an ELISA. (a) Cell titrationof total ErBb2 in SKBr3 cells. The detection range was between about1,000 cells and about 1.37 cells. (b) Table of signal/noise (S/N) ratiobetween cells and background. The signal/noise ratio was 2.71 at the1.37 cell level when the capture antibody concentration was 1 μg/ml.

FIG. 6 shows the detection of phosphorylated ErBb2 in SKBr3 cells usinga monoclonal antibody against the extracellular domain of ErbB2 as thecapture antibody and a monoclonal antibody against phosphorylated ErbB2as the detection antibody in an ELISA. (a) Cell titration ofphosphorylated ErBb2 in SKBr3 cells. The detection range was betweenabout 500 cells and about 5 cells. (b) Table of signal/noise (S/N) ratiobetween cells and background. The signal/noise ratio was 3.03 at the 5cell level when the capture antibody concentration was 1 μg/ml.

FIG. 7 shows the detection of total and phosphorylated Erk2 protein inSKBr3 cells using monoclonal antibodies against Erk2 as the captureantibody and detection antibody in an ELISA. (a) Detection of total Erk2protein using monoclonal antibodies against Erk2 as the capture antibodyand detection antibody. (b) Detection of phosphorylated Erk2 proteinusing a monoclonal antibody against Erk2 as the capture antibody and amonoclonal antibody against phosphorylated Erk2 as the detectionantibody. (c) Table of signal/noise (S/N) ratio between cells andbackground. The signal/noise ratio was about 3 at the 1.37 cell levelfor both total and phosphorylated Erk2.

FIG. 8 shows the detection of total EGFR in A431 cells using monoclonalantibodies against the extracellular domain of EGFR as the captureantibody and detection antibody in a microarray ELISA. (a) Captureantibody dilution curve based on cell numbers. The microarray ELISA hada wide dynamic range to detect EGFR in about 1-10,000 cells with variousconcentrations of capture antibody in the dilution series. (b) Celltitration curve based upon the dilution series of capture antibodyconcentrations, which showed that EGFR could be detected from one cell(arrow). (c) Table of signal/noise (S/N) ratio between cells andbackground at various capture antibody concentrations in the dilutionseries. The signal/noise ratio was 2.11 at the one cell level when thecapture antibody concentration was 0.0625 mg/ml (arrow).

FIG. 9 shows the detection of phosphorylated EGFR in A431 cells using amonoclonal antibody against the extracellular domain of EGFR as thecapture antibody and a monoclonal antibody against phosphorylated EGFRas the detection antibody in a microarray ELISA. (a) Capture antibodydilution curve based on cell numbers. The microarray ELISA had a widedynamic range to detect phosphorylated EGFR in about 1-10,000 cells withvarious concentrations of capture antibody in the dilution series. (b)Cell titration curve based upon the dilution series of capture antibodyconcentrations, which showed that phosphorylated EGFR could be detectedfrom one cell. (c) Table of signal/noise (S/N) ratio between cells andbackground at various capture antibody concentrations in the dilutionseries. The signal/noise ratio was 1.33 at the one cell level when thecapture antibody concentration was 0.125 mg/ml (arrow).

FIG. 10 shows the detection of total ErBb2 in SKBr3 cells usingmonoclonal antibodies against the extracellular domain of ErBb2 as thecapture antibody and detection antibody in a microarray ELISA. (a)Capture antibody dilution curve based on cell numbers. The microarrayELISA had a wide dynamic range to detect ErBb2 in about 1-10,000 cellswith various concentrations of capture antibody in the dilution series.(b) Cell titration curve based upon the dilution series of captureantibody concentrations, which showed that ErBb2 could be detected fromone cell (arrow). (c) Table of signal/noise (S/N) ratio between cellsand background at various capture antibody concentrations in thedilution series. The signal/noise ratio was 15.27 at the one cell levelwhen the capture antibody concentration was 0.125 mg/ml (arrow).

FIG. 11 shows the detection of phosphorylated ErBb2 in SKBr3 cells usinga monoclonal antibody against the extracellular domain of ErBb2 as thecapture antibody and a monoclonal antibody against phosphorylated ErBb2as the detection antibody in a microarray ELISA. (a) Capture antibodydilution curve based on cell numbers. The microarray ELISA had a widedynamic range to detect ErBb2 in about 1-10,000 cells with variousconcentrations of capture antibody in the dilution series. (b) Celltitration curve based upon the dilution series of capture antibodyconcentrations, which showed that phosphorylated ErBb2 could be detectedfrom one cell (arrow). (c) Table of signal/noise (S/N) ratio betweencells and background at various capture antibody concentrations in thedilution series. The signal/noise ratio was 5.45 at the one cell levelwhen the capture antibody concentration was 0.125 mg/ml (arrow).

FIG. 12 shows a comparison of the sensitivity of the proximity dualdetector microarray ELISA versus the single detector microarray ELISA.A431 cells were diluted from 10,000 to 0.01 cells. Capture antibodieswere serially diluted from 1 mg/ml to 0.004 mg/ml.

FIG. 13 shows the assay specificity for the single detector microarrayELISA versus the proximity dual detector microarray ELISA. (a) Titrationcurves of phosphorylated EGFR in A431 cells at various capture antibodyconcentrations in the single detector format. Very high background wasobserved due to the lack of specificity of the single detection antibodyin this format. (b) Titration curves of phosphorylated EGFR in A431cells at various capture antibody concentrations in the proximity dualdetector format. Very low background was observed due to the increasedspecificity obtained by detecting the proximity between two detectionantibodies in this format.

FIG. 14 shows an exemplary embodiment of the format of the addressablemicroarray using a dilution series of capture antibodies to determinethe activation states of a plurality of signal transducer molecules.

FIG. 15 shows the detection of phosphorylated Shc levels in a titrationanalysis of stimulated A431 cells. The addressable array simultaneouslyprovided information on EGFR and HER2 phosphorylation.

FIG. 16 shows the dilution curves of an anti-EGFR capture antibody. Thedynamic range of this assay was greater than 5 logs. Each individualcurve had a dynamic range of about 2 logs, but the dynamic range wassignificantly enhanced when the information from the 6 informativecurves was combined.

FIG. 17 shows a quality control procedure for the oligonucleotideconjugates of the present invention. (a) The microarray spotting patternof various GO-oligonucleotide fractions is shown. (b) The Alexa647-oligonucleotide-conjugated antibodies had the highest bindingaffinity for the GO-oligonucleotides in fractions 13-15.

FIG. 18 shows the formation of a multiplexed phosphorylated EGFR complexcomprising an Alexa 647-oligonucleotide-conjugated anti-EGFR antibodyhybridized to a glucose oxidase (GO)-oligonucleotide, an HRP-conjugatedanti-phosphorylated EGFR antibody, and an EGFR capture antibodyrestrained on a solid support.

FIG. 19 shows the simultaneous detection of total EGFR andphosphorylated EGFR. (a) Total EGFR present in 10,000 cells was detectedby the Alexa 647 signal generated from the Alexa647-oligonucleotide-conjugated anti-EGFR antibody. The degree of EGFRphosphorylation was detected by monitoring the tyramide-mediatedamplified Alexa 555 signal. (b) Total EGFR (t-EGFR) was detected by adirect binding assay from as few as 10 cells and phosphorylated EGFR(p-EGFR) was detected from 1 cell. 10e⁵ p-EGFR molecules were detectedwith the proximity signal amplification method. The detection limit ofp-EGFR was increased over 100-fold by using the proximity assay format.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The present invention provides methods for detecting the activationstate and/or total amount of a plurality of signal transductionmolecules in rare circulating cells using an antibody-based array assaysystem. In some embodiments, the multiplex, high-throughput immunoassaysof the present invention can detect the activation state of one or moresignal transduction molecules in circulating cells of a solid tumor atthe single cell level. In fact, signal transduction molecules such asEGFR can be detected with a sensitivity of about 100 zeptomoles and alinear dynamic range of from about 100 zeptomoles to about 100femtomoles. As such, single-cell detection of the activation state ofmultiple signal transducers in rare circulating cells facilitates cancerprognosis and diagnosis as well as the design of personalized, targetedtherapies.

Rare circulating cells include circulating cells of a solid tumor thathave either metastasized or micrometastasized from a solid tumor.Circulating tumor cells, cancer stem cells, and cells that are migratingto a tumor (e.g., due to chemoattraction) such as circulatingendothelial progenitor cells, circulating endothelial cells, circulatingpro-angiogenic myeloid cells, and circulating dendritic cells are someexamples of circulating cells of a solid tumor.

Signal transduction molecules of interest are typically extractedshortly after the circulating cells are isolated to preserve their insitu activation state, preferably within about 24, 6, or 1 hr, and morepreferably within about 30, 15 or 5 minutes. The isolated cells may alsobe incubated with one or more growth factors, usually at nanomolar tomicromolar concentrations, for about 1-30 minutes to resuscitate orstimulate activation of the signal transduction molecules (see, e.g.,Irish et al., Cell, 118:217-228 (2004)).

As explained in greater detail herein, to evaluate potential anticancertherapies for an individual patient, the isolated cells can be incubatedwith one or more anticancer drugs at varying doses. Growth factorstimulation can then be performed for a few minutes (e.g., about 1-5minutes) or for several hours (e.g., about 1-6 hours). The differentialactivation of signaling pathways with and without anticancer drugs canaid in the selection of a suitable cancer therapy at the proper dose foreach individual patent. Circulating cells can also be isolated from apatient sample during anticancer drug treatment and stimulated with oneor more growth factors to determine whether a change in therapy shouldbe implemented. As such, the methods of the present inventionadvantageously assist the clinician in providing the right anticancerdrug at the right dose at the right time for every patient.

II. Definitions

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

The term “cancer” is intended to include any member of a class ofdiseases characterized by the uncontrolled growth of aberrant cells. Theterm includes all known cancers and neoplastic conditions, whethercharacterized as malignant, benign, soft tissue, or solid, and cancersof all stages and grades including pre- and post-metastatic cancers.Examples of different types of cancer include, but are not limited to,lung cancer (e.g., non-small cell lung cancer); digestive andgastrointestinal cancers such as colorectal cancer, gastrointestinalstromal tumors, gastrointestinal carcinoid tumors, colon cancer, rectalcancer, anal cancer, bile duct cancer, small intestine cancer, andstomach (gastric) cancer; esophageal cancer; gallbladder cancer; livercancer; pancreatic cancer; appendix cancer; breast cancer; ovariancancer; renal cancer (e.g., renal cell carcinoma); cancer of the centralnervous system; skin cancer; lymphomas; choriocarcinomas; head and neckcancers; osteogenic sarcomas; and blood cancers. As used herein, a“tumor” comprises one or more cancerous cells.

The term “analyte” includes any molecule of interest, typically amacromolecule such as a polypeptide, whose presence, amount, and/oridentity is determined. In certain instances, the analyte is a cellularcomponent of circulating cells of a solid tumor, preferably a signaltransduction molecule.

As used herein, the term “dilution series” is intended to include aseries of descending concentrations of a particular sample (e.g., celllysate) or reagent (e.g., antibody). A dilution series is typicallyproduced by a process of mixing a measured amount of a startingconcentration of a sample or reagent with a diluent (e.g., dilutionbuffer) to create a lower concentration of the sample or reagent, andrepeating the process enough times to obtain the desired number ofserial dilutions. The sample or reagent can be serially diluted at least2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 500, or1000-fold to produce a dilution series comprising at least 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40,45, or 50 descending concentrations of the sample or reagent. Forexample, a dilution series comprising a 2-fold serial dilution of acapture antibody reagent at a 1 mg/ml starting concentration can beproduced by mixing an amount of the starting concentration of captureantibody with an equal amount of a dilution buffer to create a 0.5 mg/mlconcentration of the capture antibody, and repeating the process toobtain capture antibody concentrations of 0.25 mg/ml, 0.125 mg/ml,0.0625 mg/ml, 0.0325 mg/ml, etc.

The term “superior dynamic range” as used herein refers to the abilityof an assay of the present invention to detect a specific analyte in asfew as one cell or in as many as thousands of cells. For example, theimmunoassays described herein possess superior dynamic range becausethey advantageously detect a particular signal transduction molecule ofinterest in about 1-10,000 cells (e.g., about 1, 5, 10, 25, 50, 75, 100,250, 500, 750, 1000, 2500, 5000, 7500, or 10,000 cells) using a dilutionseries of capture antibody concentrations.

The term “signal transduction molecule” or “signal transducer” includesproteins and other molecules that carry out the process by which a cellconverts an extracellular signal or stimulus into a response, typicallyinvolving ordered sequences of biochemical reactions inside the cell.Examples of signal transduction molecules include, but are not limitedto, receptor tyrosine kinases such as EGFR (e.g., EGFR/HER1/ErbB1,HER2/Neu/ErbB2, HER3/ErbB3, HER4/ErbB4), VEGFR-1/FLT-1,VEGFR-2/FLK-1/KDR, VEGFR-3/FLT-4, FLT-3/FLK-2, PDGFR (e.g., PDGFRA,PDGFRB), c-KIT/SCFR, INSR (insulin receptor), IGF-IR, IGF-IIR, IRR(insulin receptor-related receptor), CSF-1R, FGFR 1-4, HGFR 1-2, CCK4,TRK A-C, MET, RON, EPHA 1-8, EPHB 1-6, AXL, MER, TYRO3, TIE 1-2, TEK,RYK, DDR 1-2, RET, c-ROS, LTK (leukocyte tyrosine kinase), ALK(anaplastic lymphoma kinase), ROR 1-2, MUSK, AATYK 1-3, and RTK 106;non-receptor tyrosine kinases such as BCR-ABL, Src, Frk, Btk, Csk, Abl,Zap70, Fes/Fps, Fak, Jak, Ack, and LIMK; tyrosine kinase signalingcascade components such as Akt, MAPK/ERK, MEK, RAF, PLA2, MEKK, JNKK,JNK, p38, Shc (p66), PI3K, Ras (e.g., K-Ras, N-Ras, H-Ras), Rho, Rac1,Cdc42, PLC, PKC, p70 S6 kinase, p53, cyclin D1, STAT1, STAT3, PIP2,PIP3, PDK, mTOR, BAD, p21, p27, ROCK, IP3, TSP-1, NOS, PTEN, RSK 1-3,JNK, c-Jun, Rb, CREB, Ki67, and paxillin; and combinations thereof.

As used herein, the term “circulating cells” comprises cells that haveeither metastasized or micrometastasized from a solid tumor. Examples ofcirculating cells include, but are not limited to, circulating tumorcells, cancer stem cells, and/or cells that are migrating to the tumor(e.g., circulating endothelial progenitor cells, circulating endothelialcells, circulating pro-angiogenic myeloid cells, circulating dendriticcells, etc.).

The term “sample” as used herein includes any biological specimenobtained from a patient. Samples include, without limitation, wholeblood, plasma, serum, red blood cells, white blood cells (e.g.,peripheral blood mononuclear cells), saliva, urine, stool (i.e., feces),sputum, bronchial lavage fluid, tears, nipple aspirate, lymph (e.g.,disseminated tumor cells of the lymph node), fine needle aspirate, anyother bodily fluid, a tissue sample (e.g., tumor tissue) such as abiopsy of a tumor (e.g., needle biopsy), and cellular extracts thereof.In some embodiments, the sample is whole blood or a fractional componentthereof such as plasma, serum, or a cell pellet. In preferredembodiments, the sample is obtained by isolating circulating cells of asolid tumor from whole blood or a cellular fraction thereof using anytechnique known in the art and preparing a cellular extract of thecirculating cells. In other embodiments, the sample is a formalin fixedparaffin embedded (FFPE) tumor tissue sample, e.g., from a solid tumorof the lung, colon, or rectum.

The term “subject” or “patient” typically includes humans, but can alsoinclude other animals such as, e.g., other primates, rodents, canines,felines, equines, ovines, porcines, and the like.

An “array” or “microarray” comprises a distinct set and/or dilutionseries of capture antibodies immobilized or restrained on a solidsupport such as, for example, glass (e.g., a glass slide), plastic,chips, pins, filters, beads (e.g., magnetic beads, polystyrene beads,etc.), paper, membrane (e.g., nylon, nitrocellulose, polyvinylidenefluoride (PVDF), etc.), fiber bundles, or any other suitable substrate.The capture antibodies are generally immobilized or restrained on thesolid support via covalent or noncovalent interactions (e.g., ionicbonds, hydrophobic interactions, hydrogen bonds, Van der Waals forces,dipole-dipole bonds). In certain instances, the capture antibodiescomprise capture tags which interact with capture agents bound to thesolid support. The arrays used in the assays of the present inventiontypically comprise a plurality of different capture antibodies and/orcapture antibody concentrations that are coupled to the surface of asolid support in different known/addressable locations.

The term “capture antibody” is intended to include an immobilizedantibody which is specific for (i.e., binds, is bound by, or forms acomplex with) one or more analytes of interest in a sample such as acellular extract of circulating cells of a solid tumor. In preferredembodiments, the capture antibody is restrained on a solid support in anarray. Suitable capture antibodies for immobilizing any of a variety ofsignal transduction molecules on a solid support are available fromUpstate (Temecula, Calif.), Biosource (Camarillo, Calif.), CellSignaling Technologies (Danvers, Mass.), R&D Systems (Minneapolis,Minn.), Lab Vision (Fremont, Calif.), Santa Cruz Biotechnology (SantaCruz, Calif.), Sigma (St. Louis, Mo.), and BD Biosciences (San Jose,Calif.).

The term “detection antibody” as used herein includes an antibodycomprising a detectable label which is specific for (i.e., binds, isbound by, or forms a complex with) one or more analytes of interest in asample. The term also encompasses an antibody which is specific for oneor more analytes of interest, wherein the antibody can be bound byanother species that comprises a detectable label. Examples ofdetectable labels include, but are not limited to, biotin/streptavidinlabels, nucleic acid (e.g., oligonucleotide) labels, chemically reactivelabels, fluorescent labels, enzyme labels, radioactive labels, andcombinations thereof. Suitable detection antibodies for detecting theactivation state and/or total amount of any of a variety of signaltransduction molecules are available from Upstate (Temecula, Calif.),Biosource (Camarillo, Calif.), Cell Signaling Technologies (Danvers,Mass.), R&D Systems (Minneapolis, Minn.), Lab Vision (Fremont, Calif.),Santa Cruz Biotechnology (Santa Cruz, Calif.), Sigma (St. Louis, Mo.),and BD Biosciences (San Jose, Calif.). As a non-limiting example,phospho-specific antibodies against various phosphorylated forms ofsignal transduction molecules such as EGFR, c-KIT, c-Src, FLK-1, PDGFRA,PDGFRB, Akt, MAPK, PTEN, Raf, and MEK are available from Santa CruzBiotechnology.

The term “activation state-dependent antibody” includes a detectionantibody which is specific for (i.e., binds, is bound by, or forms acomplex with) a particular activation state of one or more analytes ofinterest in a sample. In preferred embodiments, the activationstate-dependent antibody detects the phosphorylation, ubiquitination,and/or complexation state of one or more analytes such as one or moresignal transduction molecules. In some embodiments, the phosphorylationof members of the EGFR family of receptor tyrosine kinases and/or theformation of heterodimeric complexes between EGFR family members isdetected using activation state-dependent antibodies. Non-limitingexamples of activation states (listed in parentheses) that are suitablefor detection with activation state-dependent antibodies include: EGFR(EGFRvIII, phosphorylated (p-) EGFR, EGFR:Shc, ubiquitinated (u-) EGFR,p-EGFRvIII); ErbB2 (p85:truncated (Tr)-ErbB2, p-ErbB2, p85:Tr-p-ErbB2,Her2:Shc, ErbB2:PI3K, ErbB2:EGFR, ErbB2:ErbB3, ErbB2:ErbB4); ErbB3(p-ErbB3, ErbB3:PI3K, p-ErbB3:PI3K, ErbB3:Shc); ErbB4 (p-ErbB4,ErbB4:Shc); IGF-1R (p-IGF-1R, IGF-1R:IRS, IRS:PI3K, p-IRS, IGF-1R:PI3K);INSR (p-INSR); KIT (p-KIT); FLT3 (p-FLT3); HGFR1 (p-HGFR1); HGFR2(p-HGFR2); RET (p-RET); PDGFRa (p-PDGFRa); PDGFRP (p-PDGFRP); VEGFR1(p-VEGFR1, VEGFR1:PLCγ, VEGFR1:Src); VEGFR2 (p-VEGFR2, VEGFR2:PLCγ,VEGFR2:Src, VEGFR2:heparin sulfate, VEGFR2:VE-cadherin); VEGFR3(p-VEGFR3); FGFR1 (p-FGFR1); FGFR2 (p-FGFR2); FGFR3 (p-FGFR3); FGFR4(p-FGFR4); Tie1 (p-Tie1); Tie2 (p-Tie2); EphA (p-EphA); EphB (p-EphB);NFKB and/or IKB (p-IK (S32), p-NFKB (S536), p-P65:IKBa); Akt (p-Akt(T308, S473)); PTEN (p-PTEN); Bad (p-Bad (S112, S136), Bad:14-3-3); mTor(p-mTor (S2448)); p70S6K (p-p70S6K (T229, T389)); Mek (p-Mek (S217,S221)); Erk (p-Erk (T202, Y204)); Rsk-1 (p-Rsk-1 (T357, S363)); Jnk(p-Jnk (T183, Y185)); P38 (p-P38 (T180, Y182)); Stat3 (p-Stat-3 (Y705,S727)); Fak (p-Fak (Y576)); Rb (p-Rb (S249, T252, S780)); Ki67; p53(p-p53 (S392, S20)); CREB (p-CREB (S133)); c-Jun (p-c-Jun (S63)); cSrc(p-cSrc (Y416)); and paxillin (p-paxillin (Y118)).

The term “activation state-independent antibody” includes a detectionantibody which is specific for (i.e., binds, is bound by, or forms acomplex with) one or more analytes of interest in a sample irrespectiveof their activation state. For example, the activation state-independentantibody can detect both phosphorylated and unphosphorylated forms ofone or more analytes such as one or more signal transduction molecules.

The term “nucleic acid” or “polynucleotide” includesdeoxyribonucleotides or ribonucleotides and polymers thereof in eithersingle- or double-stranded form such as, for example, DNA and RNA.Nucleic acids include nucleic acids containing known nucleotide analogsor modified backbone residues or linkages, which are synthetic,naturally occurring, and non-naturally occurring, and which have similarbinding properties as the reference nucleic acid. Examples of suchanalogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2′-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unlessspecifically limited, the term encompasses nucleic acids containingknown analogues of natural nucleotides that have similar bindingproperties as the reference nucleic acid. Unless otherwise indicated, aparticular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof and complementary sequences aswell as the sequence explicitly indicated.

The term “oligonucleotide” refers to a single-stranded oligomer orpolymer of RNA, DNA, RNA/DNA hybrid, and/or a mimetic thereof. Incertain instances, oligonucleotides are composed of naturally-occurring(i.e., unmodified) nucleobases, sugars, and internucleoside (backbone)linkages. In certain other instances, oligonucleotides comprise modifiednucleobases, sugars, and/or internucleoside linkages.

As used herein, the term “mismatch motif” or “mismatch region” refers toa portion of an oligonucleotide that does not have 100% complementarityto its complementary sequence. An oligonucleotide may have at least one,two, three, four, five, six, or more mismatch regions. The mismatchregions may be contiguous or may be separated by 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, or more nucleotides. The mismatch motifs or regions maycomprise a single nucleotide or may comprise two, three, four, five, ormore nucleotides.

The phrase “stringent hybridization conditions” refers to conditionsunder which an oligonucleotide will hybridize to its complementarysequence, but to no other sequences. Stringent conditions aresequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures. Anextensive guide to the hybridization of nucleic acids is found inTijssen, Techniques in Biochemistry and Molecular Biology—Hybridizationwith Nucleic Probes, “Overview of principles of hybridization and thestrategy of nucleic acid assays” (1993). Generally, stringent conditionsare selected to be about 5-10° C. lower than the thermal melting point(T_(m)) for the specific sequence at a defined ionic strength pH. TheT_(m) is the temperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at T_(m), 50% of the probes are occupied atequilibrium). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. For selective orspecific hybridization, a positive signal is at least two timesbackground, preferably 10 times background hybridization.

The terms “substantially identical” or “substantial identity,” in thecontext of two or more nucleic acids, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides that are the same (i.e., at least about 60%, preferably atleast about 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over aspecified region) when compared and aligned for maximum correspondenceover a comparison window or designated region as measured using asequence comparison algorithm or by manual alignment and visualinspection. This definition, when the context indicates, also refersanalogously to the complement of a sequence. Preferably, the substantialidentity exists over a region that is at least about 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 75, or 100 nucleotides in length.

The term “incubating” is used synonymously with “contacting” and“exposing” and does not imply any specific time or temperaturerequirements unless otherwise indicated.

III. Description of the Embodiments

The present invention provides antibody-based arrays for detecting theactivation state and/or total amount of a plurality of signaltransduction molecules in rare circulating cells and methods of usethereof for facilitating cancer prognosis and diagnosis and the designof personalized, targeted therapies.

A. Antibody Arrays

In one aspect, the present invention provides an array having superiordynamic range comprising a plurality of dilution series of captureantibodies specific for one or more analytes in a cellular extract,wherein the capture antibodies are restrained on a solid support.

In some embodiments, the cellular extract comprises an extract ofcirculating cells of a solid tumor. The circulating cells are typicallyisolated from a patient sample using one or more separation methodsincluding, for example, immunomagnetic separation (see, e.g., Racila etal., Proc. Natl. Acad. Sci. USA, 95:4589-4594 (1998); Bilkenroth et al.,Int. J. Cancer, 92:577-582 (2001)), microfluidic separation (see, e.g.,Mohamed et al., IEEE Trans. Nanobiosci., 3:251-256 (2004); Lin et al.,Abstract No. 5147, 97th AACR Annual Meeting, Washington, D.C. (2006)),FACS (see, e.g., Mancuso et al., Blood, 97:3658-3661 (2001)), densitygradient centrifugation (see, e.g., Baker et al., Clin. Cancer Res.,13:4865-4871 (2003)), and depletion methods (see, e.g., Meye et al.,Int. J. Oncol., 21:521-530 (2002)).

In other embodiments, the patient sample comprises a whole blood, serum,plasma, urine, sputum, bronchial lavage fluid, tears, nipple aspirate,lymph, saliva, and/or fine needle aspirate sample. In certain instances,the whole blood sample is separated into a plasma or serum fraction anda cellular fraction (i.e., cell pellet). The cellular fraction typicallycontains red blood cells, white blood cells, and/or circulating cells ofa solid tumor such as circulating tumor cells (CTCs), circulatingendothelial cells (CECs), circulating endothelial progenitor cells(CEPCs), cancer stem cells (CSCs), and combinations thereof. The plasmaor serum fraction usually contains, inter alia, nucleic acids (e.g.,DNA, RNA) and proteins that are released by circulating cells of a solidtumor.

In some instances, the isolated circulating cells can be stimulated invitro with one or more growth factors before, during, and/or afterincubation with one or more anticancer drugs of interest. Stimulatorygrowth factors include, but are not limited to, epidermal growth factor(EGF), heregulin (HRG), TGF-α, PIGF, angiopoietin (Ang), NRG1, PGF,TNF-α, VEGF, PDGF, IGF, FGF, HGF, cytokines, and the like. In otherinstances, the isolated circulating cells can be lysed, e.g., followinggrowth factor stimulation and/or anticancer drug treatment, to producethe cellular extract (e.g., cell lysate) using any technique known inthe art. Preferably, the cell lysis is initiated between about 1-360minutes after growth factor stimulation, and more preferably at twodifferent time intervals: (1) at about 1-5 minutes after growth factorstimulation; and (2) between about 30-180 minutes after growth factorstimulation. Alternatively, the cell lysate can be stored at −80° C.until use.

In certain embodiments, the anticancer drug comprises an anti-signalingagent (i.e., a cytostatic drug) such as a monoclonal antibody or atyrosine kinase inhibitor; an anti-proliferative agent; achemotherapeutic agent (i.e., a cytotoxic drug); and/or any othercompound with the ability to reduce or abrogate the uncontrolled growthof aberrant cells such as cancerous cells. In some embodiments, theisolated circulating cells are treated with an anti-signaling agentand/or an anti-proliferative agent in combination with one or morechemotherapeutic agents.

Examples of anti-signaling agents suitable for use in the presentinvention include, without limitation, monoclonal antibodies such astrastuzumab (Herceptin®), alemtuzumab (Campath®), bevacizumab(Avastin®), cetuximab (Erbitux®), gemtuzumab (Mylotarg®), panitumumab(Vectibix™), rituximab (Rituxan®), and tositumomab (BEXXAR®); tyrosinekinase inhibitors such as gefitinib (Iressa®), sunitinib (Sutent®),erlotinib (Tarceva®), lapatinib (GW-572016), canertinib (CI 1033),semaxinib (SU5416), vatalanib (PTK787/ZK222584), sorafenib (BAY 43-9006;Nexavar®), imatinib mesylate (Gleevec®), and leflunomide (SU101); andcombinations thereof.

Exemplary anti-proliferative agents include mTOR inhibitors such assirolimus (rapamycin), temsirolimus (CCI-779), and everolimus (RAD001);Akt inhibitors such as1L6-hydroxymethyl-chiro-inositol-2-(R)-2-O-methyl-3-O-octadecyl-sn-glycerocarbonate,9-methoxy-2-methylellipticinium acetate,1,3-dihydro-1-(1-((4-(6-phenyl-1H-imidazo[4,5-g]quinoxalin-7-yl)phenyl)methyl)-4-piperidinyl)-2H-benzimidazol-2-one,10-(4′-(N-diethylamino)butyl)-2-chlorophenoxazine, 3-formylchromonethiosemicarbazone (Cu(II)Cl₂ complex), API-2, a 15-mer peptide derivedfrom amino acids 10-24 of the proto-oncogene TCL1 (Hiromura et al., J.Biol. Chem., 279:53407-53418 (2004), KP372-1, and the compoundsdescribed in Kozikowski et al., J. Am. Chem. Soc., 125:1144-1145 (2003)and Kau et al., Cancer Cell, 4:463-476 (2003); and combinations thereof.

Non-limiting examples of chemotherapeutic agents include platinum-baseddrugs (e.g., oxaliplatin, cisplatin, carboplatin, spiroplatin,iproplatin, satraplatin, etc.), alkylating agents (e.g.,cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan,mechlorethamine, uramustine, thiotepa, nitrosoureas, etc.),anti-metabolites (e.g., 5-fluorouracil, azathioprine, 6-mercaptopurine,methotrexate, leucovorin, capecitabine, cytarabine, floxuridine,fludarabine, gemcitabine, pemetrexed, raltitrexed, etc.), plantalkaloids (e.g., vincristine, vinblastine, vinorelbine, vindesine,podophyllotoxin, paclitaxel, docetaxel, etc.), topoisomerase inhibitors(e.g., irinotecan, topotecan, amsacrine, etoposide (VP16), etoposidephosphate, teniposide, etc.), antitumor antibiotics (e.g., doxorubicin,adriamycin, daunorubicin, epirubicin, actinomycin, bleomycin, mitomycin,mitoxantrone, plicamycin, etc.), pharmaceutically acceptable saltsthereof, stereoisomers thereof, derivatives thereof, analogs thereof,and combinations thereof.

In preferred embodiments, the one or more analytes in the cellularextract comprise a plurality of signal transduction molecules. Examplesof signal transduction molecules of interest are described above andinclude receptor tyrosine kinases, non-receptor tyrosine kinases, and/ortyrosine kinase signaling cascade components.

In some embodiments, each dilution series of capture antibodiescomprises a series of descending capture antibody concentrations. Incertain instances, the capture antibodies are serially diluted at least2-fold (e.g., 2, 5, 10, 20, 50, 100, 500, or 1000-fold) to produce adilution series comprising a set number (e.g., 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25, or more) of descending capture antibody concentrationswhich are spotted onto the array. Preferably, at least 2, 3, 4, 5, or 6replicates of each capture antibody dilution are spotted onto the array.

In other embodiments, the solid support comprises glass (e.g., a glassslide), plastic, chips, pins, filters, beads, paper, membrane (e.g.,nylon, nitrocellulose, polyvinylidene fluoride (PVDF), etc.), fiberbundles, or any other suitable substrate. In a preferred embodiment, thecapture antibodies are restrained (e.g., via covalent or noncovalentinteractions) on glass slides coated with a nitrocellulose polymer suchas, for example, FAST® Slides, which are commercially available fromWhatman Inc. (Florham Park, N.J.).

As a non-limiting example, FIG. 14 illustrates an addressable microarraycomprising a plurality of dilution series of capture antibodies todetermine the activation states of EGFR, HER2, Shc, Erk, and PI3K inwhich the capture antibodies in each dilution series are directed to oneof these analytes. Accordingly, the arrays of the present inventioncomprise a plurality of different capture antibodies in a series ofdescending concentrations (i.e., serial dilutions), wherein the captureantibodies are coupled to the surface of the solid support in differentaddressable locations.

One skilled in the art will appreciate that the array can be anyconfiguration that allows discrete signals for each of the activatedsignal transduction molecules to be detected. For example, the array canbe a line or a grid of distinct regions (e.g., dots or spots) on thesupport surface, where each region contains a different capture antibodyor capture agent (i.e., to bind the capture tag present on the captureantibody). The array can be configured for use in methods where theactivation states of a plurality of signal transduction molecules aredetected in a single, multiplex assay. In various embodiments, theplurality comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,35, 40, 45, 50, or more signal transduction molecules.

B. Single Detection Assays

In another aspect, the assay for detecting the activation state of aparticular analyte of interest in a cellular extract of tumor cells suchas circulating cells of a solid tumor is a multiplex, high-throughputsingle detection (i.e., two-antibody) assay having superior dynamicrange. As a non-limiting example, the two antibodies used in the assaycan comprise: (1) a capture antibody specific for the analyte; and (2) adetection antibody specific for an activated form of the analyte (i.e.,activation state-dependent antibody). The activation state-dependentantibody is capable of detecting, for example, the phosphorylation,ubiquitination, and/or complexation state of the analyte. Alternatively,the detection antibody comprises an activation state-independentantibody, which detects the total amount of the analyte in the cellularextract. The activation state-independent antibody is generally capableof detecting both the activated and non-activated forms of the analyte.

In a preferred aspect, the present invention provides a method forperforming a multiplex, high-throughput immunoassay having superiordynamic range, the method comprising:

-   -   (a) incubating a cellular extract with a plurality of dilution        series of capture antibodies specific for one or more analytes        in the cellular extract to form a plurality of captured        analytes, wherein the capture antibodies are restrained on a        solid support;    -   (b) incubating the plurality of captured analytes with detection        antibodies specific for the corresponding analytes to form a        plurality of detectable captured analytes;    -   (c) incubating the plurality of detectable captured analytes        with first and second members of a signal amplification pair to        generate an amplified signal; and    -   (d) detecting an amplified signal generated from the first and        second members of the signal amplification pair.

In some embodiments, the cellular extract comprises an extract ofcirculating cells of a solid tumor. The circulating cells are typicallyisolated from a patient sample using one or more separation methodsknown in the art including, for example, immunomagnetic separation,microfluidic separation, FACS, density gradient centrifugation, anddepletion methods. Those of skill in the art will know of other methodssuitable for the separation and/or isolation of circulating cells.

In other embodiments, the patient sample comprises a whole blood, serum,plasma, urine, sputum, bronchial lavage fluid, tears, nipple aspirate,lymph, saliva, and/or fine needle aspirate sample. In certain instances,the whole blood sample is separated into a plasma or serum fraction anda cellular fraction (i.e., cell pellet). The cellular fraction typicallycontains red blood cells, white blood cells, and/or circulating cells ofa solid tumor such as CTCs, CECs, CEPCs, and/or CSCs. The plasma orserum fraction usually contains, inter alia, nucleic acids (e.g., DNA,RNA) and proteins that are released by circulating cells of a solidtumor.

In some instances, the isolated circulating cells can be stimulated invitro with one or more growth factors before, during, and/or afterincubation with one or more anticancer drugs of interest. Stimulatorygrowth factors are described above. In other instances, the isolatedcirculating cells can be lysed, e.g., following growth factorstimulation and/or anticancer drug treatment, to produce the cellularextract (e.g., cell lysate) using any technique known in the art.Preferably, the cell lysis is initiated between about 1-360 minutesafter growth factor stimulation, and more preferably at two differenttime intervals: (1) at about 1-5 minutes after growth factorstimulation; and (2) between about 30-180 minutes after growth factorstimulation. Alternatively, the cell lysate can be stored at −80° C.until use.

In certain embodiments, the anticancer drug comprises an anti-signalingagent (e.g., monoclonal antibody, tyrosine kinase inhibitor, etc.), ananti-proliferative agent, a chemotherapeutic agent, and/or any othercompound with the ability to reduce or abrogate the uncontrolled growthof aberrant cells such as cancerous cells. Examples of specificanticancer drugs which fall into these general classes of therapeuticagents are provided above.

In preferred embodiments, the one or more analytes in the cellularextract comprise a plurality of signal transduction molecules. Examplesof signal transduction molecules of interest are described above andinclude, without limitation, receptor tyrosine kinases, non-receptortyrosine kinases, and/or tyrosine kinase signaling cascade components.

In some embodiments, each dilution series of capture antibodiescomprises a series of descending capture antibody concentrations. Incertain instances, the capture antibodies are serially diluted at least2-fold (e.g., 2, 5, 10, 20, 50, 100, 500, or 1000-fold) to produce adilution series comprising a set number (e.g., 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25, or more) of descending capture antibody concentrationswhich are spotted onto the array. Preferably, at least 2, 3, 4, 5, or 6replicates of each capture antibody dilution are spotted onto the array.

In other embodiments, the solid support comprises glass (e.g., a glassslide), plastic, chips, pins, filters, beads, paper, membrane (e.g.,nylon, nitrocellulose, PVDF, etc.), fiber bundles, or any other suitablesubstrate. In a preferred embodiment, the capture antibodies arerestrained on glass slides coated with a nitrocellulose polymer such as,for example, FAST® Slides (Whatman Inc.; Florham Park, N.J.).

In certain instances, the cellular extract is incubated with captureantibodies already restrained on a solid support. In certain otherinstances, the cellular extract is first incubated with captureantibodies in solution and then contacted with a solid support toimmobilize the captured analytes, e.g., via capture tags present on thecapture antibodies which interact with capture agents bound to the solidsupport.

In some embodiments, the detection antibodies are incubated withanalytes that are bound to capture antibodies in solution or restrainedon a solid support. In certain instances, the cellular extractcomprising a plurality of analytes is first incubated with the detectionantibodies in solution and then contacted with capture antibodies insolution or restrained on a solid support. In certain other instances,the cellular extract comprising a plurality of analytes is firstincubated with capture antibodies and detection antibodies in solutionand then contacted with a solid support to immobilize theantibody-analyte complexes, e.g., via capture tags present on thecapture antibodies or detection antibodies which interact with captureagents bound to the solid support.

In certain instances, the detection antibodies comprise activationstate-independent antibodies, which are useful for detecting the totalamount of one or more of the analytes in the cellular extract. As anon-limiting example, activation state-independent antibodies can detectboth phosphorylated and unphosphorylated forms of one or more signaltransduction molecules. In certain other instances, the detectionantibodies comprise activation state-dependent antibodies, which areuseful for detecting the activation state of one or more of the analytesin the cellular extract. Preferably, activation state-dependentantibodies detect the phosphorylation, ubiquitination, and/orcomplexation state of one or more signal transduction molecules.

The capture antibodies and detection antibodies are typically selectedto minimize competition between them with respect to analyte binding(i.e., both capture and detection antibodies can simultaneously bindtheir corresponding signal transduction molecules).

In a preferred embodiment, the detection antibodies comprise a firstmember of a binding pair (e.g., biotin) and the first member of thesignal amplification pair comprises a second member of the binding pair(e.g., streptavidin). The binding pair members can be coupled directlyor indirectly to the detection antibodies or to the first member of thesignal amplification pair using methods well-known in the art. Incertain instances, the first member of the signal amplification pair isa peroxidase (e.g., horseradish peroxidase (HRP), catalase,chloroperoxidase, cytochrome c peroxidase, eosinophil peroxidase,glutathione peroxidase, lactoperoxidase, myeloperoxidase, thyroidperoxidase, deiodinase, etc.), and the second member of the signalamplification pair is a tyramide reagent (e.g., biotin-tyramide). Inthese instances, the amplified signal is generated by peroxidaseoxidization of the tyramide reagent to produce an activated tyramide inthe presence of hydrogen peroxide (H₂O₂).

The activated tyramide is either directly detected or detected upon theaddition of a signal-detecting reagent such as, for example, astreptavidin-labeled fluorophore or a combination of astreptavidin-labeled peroxidase and a chromogenic reagent. Examples offluorophores suitable for use in the present invention include, but arenot limited to, an Alexa Fluor® dye (e.g., Alexa Fluor® 555),fluorescein, fluorescein isothiocyanate (FITC), Oregon Green™;rhodamine, Texas red, tetrarhodamine isothiocynate (TRITC), a CyDye™fluor (e.g., Cy2, Cy3, Cy5), and the like. The streptavidin label can becoupled directly or indirectly to the fluorophore or peroxidase usingmethods well-known in the art. Non-limiting examples of chromogenicreagents suitable for use in the present invention include3,3′,5,5′-tetramethylbenzidine (TMB), 3,3′-diaminobenzidine (DAB),2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS),4-chloro-1-napthol (4CN), and/or porphyrinogen.

One skilled in the art will appreciate that binding partners other thanantibodies can be used to immobilize and/or detect one or more analytesfrom a cellular extract in accordance with the single detection assaysdescribed herein. Non-limiting examples of such binding partners includeligands or receptors of the analyte, substrates of the analyte, bindingdomains (e.g., PTB, SH2, etc.), aptamers, and the like.

C. Proximity Dual Detection Assays

In yet another aspect, the assay for detecting the activation state of aparticular analyte of interest in a cellular extract of tumor cells suchas circulating cells of a solid tumor is a multiplex, high-throughputproximity (i.e., three-antibody) assay having superior dynamic range. Asa non-limiting example, the three antibodies used in the proximity assaycan comprise: (1) a capture antibody specific for the analyte; (2) adetection antibody specific for an activated form of the analyte (i.e.,activation state-dependent antibody); and (3) a detection antibody whichdetects the total amount of the analyte (i.e., activationstate-independent antibody). The activation state-dependent antibody iscapable of detecting, for example, the phosphorylation, ubiquitination,and/or complexation state of the analyte. The activation state-dependentantibody is generally capable of detecting both the activated andnon-activated forms of the analyte.

In a preferred aspect, the present invention provides a method forperforming a multiplex, high-throughput immunoassay having superiordynamic range, the method comprising:

-   -   (a) incubating a cellular extract with a plurality of dilution        series of capture antibodies specific for one or more analytes        in the cellular extract to form a plurality of captured        analytes, wherein the capture antibodies are restrained on a        solid support;    -   (b) incubating the plurality of captured analytes with detection        antibodies specific for the corresponding analytes to form a        plurality of detectable captured analytes, wherein the detection        antibodies comprise:        -   (1) a plurality of activation state-independent antibodies            labeled with a facilitating moiety, and        -   (2) a plurality of activation state-dependent antibodies            labeled with a first member of a signal amplification pair,        -   wherein the facilitating moiety generates an oxidizing agent            which channels to and reacts with the first member of the            signal amplification pair;    -   (c) incubating the plurality of detectable captured analytes        with a second member of the signal amplification pair to        generate an amplified signal; and    -   (d) detecting the amplified signal generated from the first and        second members of the signal amplification pair.

FIG. 1 illustrates an exemplary proximity assay in which an analyte isbound to a capture antibody and two detection antibodies (i.e., anactivation state-independent antibody and an activation state-dependentantibody). The capture antibody 1 and the activation state-independentantibody 2 each bind the analyte 6 independent of its activation state.The activation state-dependent antibody 3 binds the analyte dependent ofits activation state (e.g., the activation state-dependent antibody willonly bind an activated form of the analyte having a phosphorylatedresidue). The activation state-independent antibody is labeled with afacilitating moiety (designated M1, 4) and the activationstate-dependent antibody is labeled with a first member of a signalamplification pair (designated M2, 5). Binding of both detectionantibodies to the analyte brings the facilitating moiety withinsufficient proximity (depicted by the area inside the dotted line 7) tothe first member of the signal amplification pair such that a signalgenerated by the facilitating moiety can channel to the first member ofthe signal amplification pair resulting in the generation of adetectable and/or amplifiable signal. Various methods for proximitychanneling are known in the art and include, for example, FRET,time-resolved fluorescence-FRET, LOCI, etc. An advantage of proximitychanneling, as used in the methods of the present invention, is that asingle detectable signal is generated for only those analytes that havebound all three antibodies, resulting in increased assay specificity,lower background, and simplified detection.

In some embodiments, the cellular extract comprises an extract ofcirculating cells of a solid tumor. The circulating cells are typicallyisolated from a patient sample using one or more separation methodsknown in the art including, for example, immunomagnetic separation,microfluidic separation, FACS, density gradient centrifugation, anddepletion methods.

In other embodiments, the patient sample comprises a whole blood, serum,plasma, urine, sputum, bronchial lavage fluid, tears, nipple aspirate,lymph, saliva, and/or fine needle aspirate sample. In certain instances,the whole blood sample is separated into a plasma or serum fraction anda cellular fraction (i.e., cell pellet). The cellular fraction typicallycontains red blood cells, white blood cells, and/or circulating cells ofa solid tumor such as CTCs, CECs, CEPCs, and/or CSCs. The plasma orserum fraction usually contains, inter alia, nucleic acids (e.g., DNA,RNA) and proteins that are released by circulating cells of a solidtumor.

In some instances, the isolated circulating cells can be stimulated invitro with one or more growth factors before, during, and/or afterincubation with one or more anticancer drugs of interest. Stimulatorygrowth factors are described above. In other instances, the isolatedcirculating cells can be lysed, e.g., following growth factorstimulation and/or anticancer drug treatment, to produce the cellularextract (e.g., cell lysate) using any technique known in the art.Preferably, the cell lysis is initiated between about 1-360 minutesafter growth factor stimulation, and more preferably at two differenttime intervals: (1) at about 1-5 minutes after growth factorstimulation; and (2) between about 30-180 minutes after growth factorstimulation. Alternatively, the cell lysate can be stored at −80° C.until use.

In certain embodiments, the anticancer drug comprises an anti-signalingagent (e.g., monoclonal antibody, tyrosine kinase inhibitor, etc.), ananti-proliferative agent, a chemotherapeutic agent, and/or any othercompound with the ability to reduce or abrogate the uncontrolled growthof aberrant cells such as cancerous cells. Examples of specificanticancer drugs which fall into these general classes of therapeuticagents are provided above.

In preferred embodiments, the one or more analytes in the cellularextract comprise a plurality of signal transduction molecules. Examplesof signal transduction molecules of interest are described above andinclude, without limitation, receptor tyrosine kinases, non-receptortyrosine kinases, and/or tyrosine kinase signaling cascade components.

In some embodiments, each dilution series of capture antibodiescomprises a series of descending capture antibody concentrations. Incertain instances, the capture antibodies are serially diluted at least2-fold (e.g., 2, 5, 10, 20, 50, 100, 500, or 1000-fold) to produce adilution series comprising a set number (e.g., 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25, or more) of descending capture antibody concentrationswhich are spotted onto the array. Preferably, at least 2, 3, 4, 5, or 6replicates of each capture antibody dilution are spotted onto the array.

In other embodiments, the solid support comprises glass (e.g., a glassslide), plastic, chips, pins, filters, beads, paper, membrane (e.g.,nylon, nitrocellulose, PVDF, etc.), fiber bundles, or any other suitablesubstrate. In a preferred embodiment, the capture antibodies arerestrained on glass slides coated with a nitrocellulose polymer such as,for example, FAST® Slides (Whatman Inc.; Florham Park, N.J.).

In certain instances, the cellular extract is incubated with captureantibodies already restrained on a solid support. In certain otherinstances, the cellular extract is first incubated with captureantibodies in solution and then contacted with a solid support toimmobilize the captured analytes, e.g., via capture tags present on thecapture antibodies which interact with capture agents bound to the solidsupport.

In some embodiments, the detection antibodies are incubated withanalytes that are bound to capture antibodies in solution or restrainedon a solid support. In certain instances, the cellular extractcomprising a plurality of analytes is first incubated with the detectionantibodies in solution and then contacted with capture antibodies insolution or restrained on a solid support. In certain other instances,the cellular extract comprising a plurality of analytes is firstincubated with capture antibodies and detection antibodies in solutionand then contacted with a solid support to immobilize theantibody-analyte complexes, e.g., via capture tags present on thecapture antibodies or detection antibodies which interact with captureagents bound to the solid support. Prior to the detecting step, theimmobilized complexes can be washed to remove uncomplexed antibodies,the washed complexes can be sequentially released from the supportsurface, and proximity channeling for each of the analytes being assayedcan be detected by a suitable method as described herein.

In embodiments where the support surface comprises capture agentsrestrained in an array, the incubating step can comprise contacting thecellular extract comprising a plurality of analytes in solution with thecapture antibodies and detection antibodies, using an excess of allthree antibodies to drive the reaction to completion. In one variationof the method, the resulting antibody-analyte complexes are attached toa solid phase and washed to remove unbound antibodies. Referring to FIG.2, the capture antibody 1 can comprise a capture tag 10. The complexesare attached to a solid phase 12 via a capture agent 11 that is adheredto the solid phase and binds the capture tag, thereby immobilizing thecomplex. The immobilized complex is washed with a suitable buffer, andthen released from the solid phase by the addition of a releasing agent13. The releasing agent may function by any mechanism that results inthe release of the washed complex. In one embodiment, the capture tagcomprises a cleavable site that is recognized and cleaved by thereleasing agent. In another embodiment, depicted in FIG. 2, thereleasing agent competes with the capture tag for binding to the captureagent. For example, the capture agent may be a first oligonucleotidethat hybridizes with a partially complementary oligonucleotide (i.e.,the capture tag) attached to the capture antibody; and the releasingagent may be an oligonucleotide that is fully complementary to thecapture agent, resulting in strand displacement and release of thewashed complex from the solid phase. Other examples of suitable capturetags/capture agents/releasing agents that can be used include, but arenot limited to, 2,4-dinitrophenol (DNP)/anti-DNP antibody/2,4-DNPlysine; T2/anti-T3 antibody/T3; ouabain/anti-digoxin antibody/digoxin;and dethiobiotin/streptavidin/biotin (see, e.g., Ishikawa et al., J.Clin. Lab Anal., 12:98-107 (1998)).

After the washed complex is released from the solid phase, it is either:(1) contacted with a support surface comprising capture moleculesrestrained in an array that specifically bind capture tags on thecapture antibody, or (2) dissociated, and the dissociated detectionantibodies are contacted with a support surface comprising captureagents that specifically bind capture tags on the detection antibodies.FIG. 2 depicts the embodiment where the washed complex is dissociatedand the dissociated detection antibodies are contacted with the supportsurface 14. The support surface comprises a plurality of capturemolecules restrained in an “addressable” or “zip code” array. Eachdistinct region of the array comprises a unique capture agent 9 thatspecifically binds the capture tag 8 present on the activationstate-independent detection antibody 2 or the activation state-dependentantibody 3, thereby restraining and organizing the tagged detectionantibodies in the array. In a preferred embodiment, the capture agentsand capture tags are oligonucleotides that specifically hybridize toeach other. Addressable arrays comprising oligonucleotide capturemolecules are well known in the art (see, e.g., Keramas et al, Lab Chip,4:152-158 (2004); Delrio-Lafreniere et al., Diag. Microbiol. Infect.Dis., 48:23-31 (2004)).

The presence of the detection antibodies at each distinct region of thearray can be directly or indirectly detected with a moiety such as afacilitating moiety (designated M1, 4) or a first member of a signalamplification pair (designated M2, 5). Examples of moieties that can bedirectly detected include fluorophores, chromophores, colloidal gold,colored latex, etc. In one embodiment, the both moieties areindependently selected fluorophores. Any pair of fluorophores thatprovide a distinguishable readout while in close proximity to each othercan be used, such as, for example, Cy3/Cy5, Cy5/phycoerthrin, and thelike. Alternatively, if an oligonucleotide addressable array is used,both moieties can be the same fluorophore delivered to different zipcodes. Laser scanning confocal microscopy can be used to detectfluorophore moieties that are adhered on the array. In assays where thecomplexes are released from the array prior to detection, such as instrand displacement assays, suitable methods for detecting thefluorophore moieties include capillary flow confocal laser inducedfluorescence, nano-HPLC, micro-capillary electrophoresis, etc.

In some embodiments, the activation state-independent antibodies furthercomprise a detectable moiety. In such instances, the amount of thedetectable moiety is correlative to the amount of one or more of theanalytes in the cellular extract. Examples of detectable moietiesinclude, but are not limited to, fluorescent labels, chemically reactivelabels, enzyme labels, radioactive labels, and the like. Preferably, thedetectable moiety is a fluorophore such as an Alexa Fluor® dye (e.g.,Alexa Fluor® 647), fluorescein, fluorescein isothiocyanate (FITC),Oregon Green™; rhodamine, Texas red, tetrarhodamine isothiocynate(TRITC), a CyDye™ fluor (e.g., Cy2, Cy3, Cy5), and the like. Thedetectable moiety can be coupled directly or indirectly to theactivation state-independent antibodies using methods well known in theart.

In certain instances, the activation state-independent antibodies aredirectly labeled with the facilitating moiety. The facilitating moietycan be coupled to the activation state-independent antibodies usingmethods well-known in the art. A suitable facilitating moiety for use inthe present invention includes any molecule capable of generating anoxidizing agent which channels to (i.e., is directed to) and reacts with(i.e., binds, is bound by, or forms a complex with) another molecule inproximity (i.e., spatially near or close) to the facilitating moiety.Examples of facilitating moieties include, without limitation, enzymessuch as glucose oxidase or any other enzyme that catalyzes anoxidation/reduction reaction involving molecular oxygen (O₂) as theelectron acceptor, and photosensitizers such as methylene blue, rosebengal, porphyrins, squarate dyes, phthalocyanines, and the like.Non-limiting examples of oxidizing agents include hydrogen peroxide(H₂O₂), a singlet oxygen, and any other compound that transfers oxygenatoms or gains electrons in an oxidation/reduction reaction. Preferably,in the presence of a suitable substrate (e.g., glucose, light, etc.),the facilitating moiety (e.g., glucose oxidase, photosensitizer, etc.)generates an oxidizing agent (e.g., hydrogen peroxide (H₂O₂), singleoxygen, etc.) which channels to and reacts with the first member of thesignal amplification pair (e.g., horseradish peroxidase (HRP), haptenprotected by a protecting group, an enzyme inactivated by thioetherlinkage to an enzyme inhibitor, etc.) when the two moieties are inproximity to each other.

In certain other instances, the activation state-independent antibodiesare indirectly labeled with the facilitating moiety via hybridizationbetween an oligonucleotide linker conjugated to the activationstate-independent antibodies and a complementary oligonucleotide linkerconjugated to the facilitating moiety. The oligonucleotide linkers canbe coupled to the facilitating moiety or to the activationstate-independent antibodies using methods well-known in the art. Insome embodiments, the oligonucleotide linker conjugated to thefacilitating moiety has 100% complementarity to the oligonucleotidelinker conjugated to the activation state-independent antibodies. Inother embodiments, the oligonucleotide linker pair comprises at leastone, two, three, four, five, six, or more mismatch regions, e.g., uponhybridization under stringent hybridization conditions. One skilled inthe art will appreciate that activation state-independent antibodiesspecific for different analytes can either be conjugated to the sameoligonucleotide linker or to different oligonucleotide linkers.

The length of the oligonucleotide linkers that are conjugated to thefacilitating moiety or to the activation state-independent antibodiescan vary. In general, the linker sequence can be at least about 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 75, or 100 nucleotides in length.Typically, random nucleic acid sequences are generated for coupling. Asa non-limiting example, a library of oligonucleotide linkers can bedesigned to have three distinct contiguous domains: a spacer domain;signature domain; and conjugation domain. Preferably, theoligonucleotide linkers are designed for efficient coupling withoutdestroying the function of the facilitating moiety or activationstate-independent antibodies to which they are conjugated.

The oligonucleotide linker sequences can be designed to prevent orminimize any secondary structure formation under a variety of assayconditions. Melting temperatures are typically carefully monitored foreach segment within the linker to allow their participation in theoverall assay procedures. Generally, the range of melting temperaturesof the segment of the linker sequence is no greater than 5° C. Computeralgorithms (e.g., OLIGO 6.0) for determining the melting temperature,secondary structure, and hairpin structure under defined ionicconcentrations can be used to analyze each of the three differentdomains within each linker. The overall combined sequences can also beanalyzed for their structural characterization and their comparabilityto other conjugated oligonucleotide linker sequences, e.g., whether theywill hybridize under stringent hybridization conditions to acomplementary oligonucleotide linker.

The spacer region of the oligonucleotide linker provides adequateseparation of the conjugation domain from the oligonucleotidecrosslinking site. The conjugation domain functions to link moleculeslabeled with a complementary oligonucleotide linker sequence to theconjugation domain via nucleic acid hybridization. The nucleicacid-mediated hybridization can be performed either before or afterantibody-analyte (i.e., antigen) complex formation, providing a moreflexible assay format. Unlike many direct antibody conjugation methods,linking relatively small oligonucleotides to antibodies or othermolecules has minimal impact on the specific affinity of antibodiestowards their target analyte or on the function of the conjugatedmolecules.

In some embodiments, the signature sequence domain of theoligonucleotide linker can be used in complex multiplexed proteinassays. Multiple antibodies can be conjugated with oligonucleotidelinkers with different signature sequences. In multiplex immunoassays,reporter oligonucleotide sequences labeled with appropriate probes canbe used to detect cross-hybridization between antibodies and theirantigens in the multiplex assay format.

Oligonucleotide linkers can be conjugated to antibodies or othermolecules using several different methods. For example, oligonucleotidelinkers can be synthesized with a thiol group on either the 5′ or 3′end. The thiol group can be deprotected using reducing agents (e.g.,TCEP-HCl) and the resulting linkers can be purified by using a desaltingspin column. The resulting deprotected oligonucleotide linkers can beconjugated to the primary amines of antibodies or other types ofproteins using heterobifunctional cross linkers such as SMCC.Alternatively, 5′-phosphate groups on oligonucleotides can be treatedwith water-soluble carbodiimide EDC to form phosphate esters andsubsequently coupled to amine-containing molecules. In certaininstances, the diol on the 3′-ribose residue can be oxidized to aldehydegroups and then conjugated to the amine groups of antibodies or othertypes of proteins using reductive amination. In certain other instances,the oligonucleotide linker can be synthesized with a biotin modificationon either the 3′ or 5′ end and conjugated to streptavidin-labeledmolecules.

Oligonucleotide linkers can be synthesized using any of a variety oftechniques known in the art, such as those described in Usman et al., J.Am. Chem. Soc., 109:7845 (1987); Scaringe et al., Nucl. Acids Res.,18:5433 (1990); Wincott et al., Nucl. Acids Res., 23:2677-2684 (1995);and Wincott et al., Methods Mol. Bio., 74:59 (1997). In general, thesynthesis of oligonucleotides makes use of common nucleic acidprotecting and coupling groups, such as dimethoxytrityl at the 5′-endand phosphoramidites at the 3′-end. Suitable reagents foroligonucleotide synthesis, methods for nucleic acid deprotection, andmethods for nucleic acid purification are known to those of skill in theart.

In certain instances, the activation state-dependent antibodies aredirectly labeled with the first member of the signal amplification pair.The signal amplification pair member can be coupled to the activationstate-dependent antibodies using methods well-known in the art. Incertain other instances, the activation state-dependent antibodies areindirectly labeled with the first member of the signal amplificationpair via binding between a first member of a binding pair conjugated tothe activation state-dependent antibodies and a second member of thebinding pair conjugated to the first member of the signal amplificationpair. The binding pair members (e.g., biotin/streptavidin) can becoupled to the signal amplification pair member or to the activationstate-dependent antibodies using methods well-known in the art. Examplesof signal amplification pair members include, but are not limited to,peroxidases such horseradish peroxidase (HRP), catalase,chloroperoxidase, cytochrome c peroxidase, eosinophil peroxidase,glutathione peroxidase, lactoperoxidase, myeloperoxidase, thyroidperoxidase, deiodinase, and the like. Other examples of signalamplification pair members include haptens protected by a protectinggroup and enzymes inactivated by thioether linkage to an enzymeinhibitor.

The capture antibodies, activation state-independent antibodies, andactivation state-dependent antibodies are typically selected to minimizecompetition between them with respect to analyte binding (i.e., allantibodies can simultaneously bind their corresponding signaltransduction molecules).

In one example of proximity channeling, the facilitating moiety isglucose oxidase (GO) and the first member of the signal amplificationpair is horseradish peroxidase (HRP). When the GO is contacted with asubstrate such as glucose, it generates an oxidizing agent (i.e.,hydrogen peroxide (H₂O₂)). If the HRP is within channeling proximity tothe GO, the H₂O₂ generated by the GO is channeled to and complexes withthe HRP to form an HRP-H₂O₂ complex, which, in the presence of thesecond member of the signal amplification pair (e.g., a chemiluminescentsubstrate such as luminol or isoluminol or a fluorogenic substrate suchas tyramide (e.g., biotin-tyramide), homovanillic acid, or4-hydroxyphenyl acetic acid), generates an amplified signal. Methods ofusing GO and IRP in a proximity assay are described in, e.g., Langry etal., U.S. Dept. of Energy Report No. UCRL-ID-136797 (1999). Whenbiotin-tyramide is used as the second member of the signal amplificationpair, the HRP-H₂O₂ complex oxidizes the tyramide to generate a reactivetyramide radical that covalently binds nearby nucleophilic residues. Theactivated tyramide is either directly detected or detected upon theaddition of a signal-detecting reagent such as, for example, astreptavidin-labeled fluorophore or a combination of astreptavidin-labeled peroxidase and a chromogenic reagent. Examples offluorophores suitable for use in the present invention include, but arenot limited to, an Alexa Fluor® dye (e.g., Alexa Fluor® 555),fluorescein, fluorescein isothiocyanate (FITC), Oregon Green™;rhodamine, Texas red, tetrarhodamine isothiocynate (TRITC), a CyDye™fluor (e.g., Cy2, Cy3, Cy5), and the like. The streptavidin label can becoupled directly or indirectly to the fluorophore or peroxidase usingmethods well-known in the art. Non-limiting examples of chromogenicreagents suitable for use in the present invention include3,3′,5,5′-tetramethylbenzidine (TMB), 3,3′-diaminobenzidine (DAB),2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS),4-chloro-1-napthol (4CN), and/or porphyrinogen.

In another example of proximity channeling, the facilitating moiety is aphotosensitizer and the first member of the signal amplification pair isa large molecule labeled with multiple haptens that are protected withprotecting groups that prevent binding of the haptens to a specificbinding partner (e.g., ligand, antibody, etc.). For example, the signalamplification pair member can be a dextran molecule labeled withprotected biotin, coumarin, and/or fluorescein molecules. Suitableprotecting groups include, but are not limited to, phenoxy-, analino-,olefin-, thioether-, and selenoether-protecting groups. Additionalphotosensitizers and protected hapten molecules suitable for use in theproximity assays of the present invention are described in U.S. Pat. No.5,807,675. When the photosensitizer is excited with light, it generatesan oxidizing agent (i.e., singlet oxygen). If the hapten molecules arewithin channeling proximity to the photosensitizer, the singlet oxygengenerated by the photosensitizer is channeled to and reacts withthioethers on the protecting groups of the haptens to yield carbonylgroups (ketones or aldehydes) and sulphinic acid, releasing theprotecting groups from the haptens. The unprotected haptens are thenavailable to specifically bind to the second member of the signalamplification pair (e.g., a specific binding partner that can generate adetectable signal). For example, when the hapten is biotin, the specificbinding partner can be an enzyme-labeled streptavidin. Exemplary enzymesinclude alkaline phosphatase, β-galactosidase, HRP, etc. After washingto remove unbound reagents, the detectable signal can be generated byadding a detectable (e.g., fluorescent, chemiluminescent, chromogenic,etc.) substrate of the enzyme and detected using suitable methods andinstrumentation known in the art. Alternatively, the detectable signalcan be amplified using tyramide signal amplification and the activatedtyramide either directly detected or detected upon the addition of asignal-detecting reagent as described above.

In yet another example of proximity channeling, the facilitating moietyis a photosensitizer and the first member of the signal amplificationpair is an enzyme-inhibitor complex. The enzyme and inhibitor (e.g.,phosphonic acid-labeled dextran) are linked together by a cleavablelinker (e.g., thioether). When the photosensitizer is excited withlight, it generates an oxidizing agent (i.e., singlet oxygen). If theenzyme-inhibitor complex is within channeling proximity to thephotosensitizer, the singlet oxygen generated by the photosensitizer ischanneled to and reacts with the cleavable linker, releasing theinhibitor from the enzyme, thereby activating the enzyme. An enzymesubstrate is added to generate a detectable signal, or alternatively, anamplification reagent is added to generate an amplified signal.

In a further example of proximity channeling, the facilitating moiety isHRP, the first member of the signal amplification pair is a protectedhapten or an enzyme-inhibitor complex as described above, and theprotecting groups comprise p-alkoxy phenol. The addition ofphenylenediamine and H₂O₂ generates a reactive phenylene diimine whichchannels to the protected hapten or the enzyme-inhibitor complex andreacts with p-alkoxy phenol protecting groups to yield exposed haptensor a reactive enzyme. The amplified signal is generated and detected asdescribed above (see, e.g., U.S. Pat. Nos. 5,532,138 and 5,445,944).

One skilled in the art will appreciate that binding partners other thanantibodies can be used to immobilize and/or detect one or more analytesfrom a cellular extract in accordance with the proximity (i.e.,three-antibody) assays described herein. Non-limiting examples of suchbinding partners include ligands or receptors of the analyte, substratesof the analyte, binding domains (e.g., PTB, SH2, etc.), aptamers, andthe like.

D. Kits

In a further aspect, the present invention provides kits for performingthe antibody-based array methods described above comprising: (a) adilution series of a plurality of capture antibodies restrained on asolid support; and (b) a plurality of detection antibodies (e.g.,activation state-independent antibodies and/or activationstate-dependent antibodies). In some instances, the kits can furthercontain instructions for methods of using the kit to detect theactivation states of a plurality of signal transduction molecules ofcirculating cells of a solid tumor. The kits may also contain any of theadditional reagents described above with respect to performing thespecific methods of the present invention such as, for example, firstand second members of the signal amplification pair, tyramide signalamplification reagents, substrates for the facilitating moiety, washbuffers, capture/release reagents, etc.

IV. Construction of Antibody Arrays

In certain aspects, the present invention provides antibody-based arraysfor detecting the activation state of a plurality of signal transductionmolecules in a cellular extract of circulating cells of a solid tumorusing a dilution series of capture antibodies restrained on a solidsupport. The arrays used in the assays of the present inventiontypically comprise a plurality of different capture antibodies at arange of capture antibody concentrations that are coupled to the surfaceof a solid support in different addressable locations.

The solid support can comprise any suitable substrate for immobilizingproteins. Examples of solid supports include, but are not limited to,glass (e.g., a glass slide), plastic, chips, pins, filters, beads (e.g.,magnetic beads, polystyrene beads, etc.), paper, membranes, fiberbundles, gels, metal, ceramics, and the like. Membranes such nylon(Biotrans™, ICN Biomedicals, Inc. (Costa Mesa, Calif.); Zeta-Probe®,Bio-Rad Laboratories (Hercules, Calif.)), nitrocellulose (Protran®,Whatman Inc. (Florham Park, N.J.)), and PVDF (Immobilon™, MilliporeCorp. (Billerica, Mass.)) are suitable for use as solid supports in thearrays of the present invention. Preferably, the capture antibodies arerestrained on glass slides coated with a nitrocellulose polymer, e.g.,FAST® Slides, which are commercially available from Whatman Inc.(Florham Park, N.J.).

Particular aspects of the solid support which are desirable include theability to bind large amounts of capture antibodies, the ability to bindcapture antibodies with minimal denaturation, and the inability to bindother proteins. Another suitable aspect is that the solid supportdisplays minimal “wicking” when antibody solutions containing captureantibodies are applied to the support. A solid support with minimalwicking allows small aliquots of capture antibody solution applied tothe support to result in small, defined spots of immobilized captureantibody.

The capture antibodies are typically directly or indirectly (e.g., viacapture tags) restrained on the solid support via covalent ornoncovalent interactions (e.g., ionic bonds, hydrophobic interactions,hydrogen bonds, Van der Waals forces, dipole-dipole bonds). In someembodiments, the capture antibodies are covalently attached to the solidsupport using a homobifunctional or heterobifunctional crosslinker usingstandard crosslinking methods and conditions. Suitable crosslinkers arecommercially available from vendors such as, e.g., Pierce Biotechnology(Rockford, Ill.).

Methods for generating the arrays of the present invention include, butare not limited to, any technique used to construct protein or nucleicacid arrays. In some embodiments, the capture antibodies are spottedonto an array using a microspotter, which are typically robotic printersequipped with split pins, blunt pins, or ink jet printing. Suitablerobotic systems for printing the antibody arrays described hereininclude the PixSys 5000 robot (Cartesian Technologies; Irvine, Calif.)with ChipMaker2 split pins (TeleChem International; Sunnyvale, Calif.)as well as other robotic printers available from BioRobics (Woburn,Mass.) and Packard Instrument Co. (Meriden, Conn.). Preferably, at least2, 3, 4, 5, or 6 replicates of each capture antibody dilution arespotted onto the array.

Another method for generating the antibody arrays of the presentinvention comprises dispensing a known volume of a capture antibodydilution at each selected array position by contacting a capillarydispenser onto a solid support under conditions effective to draw adefined volume of liquid onto the support, wherein this process isrepeated using selected capture antibody dilutions at each selectedarray position to create a complete array. The method may be practicedin forming a plurality of such arrays, where the solution-depositingstep is applied to a selected position on each of a plurality of solidsupports at each repeat cycle. A further description of such a methodcan be found, e.g., in U.S. Pat. No. 5,807,522.

In certain instances, devices for printing on paper can be used togenerate the antibody arrays of the present invention. For example, thedesired capture antibody dilution can be loaded into the printhead of adesktop jet printer and printed onto a suitable solid support (see,e.g., Silzel et al., Clin. Chem., 44:2036-2043 (1998)).

In some embodiments, the array generated on the solid support has adensity of at least about 5 spots/cm², and preferably at least about 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170,180, 190, 200, 210, 220, 230, 250, 275, 300, 325, 350, 375, 400, 425,450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2000,3000, 4000, 5000, 6000, 7000, 8000 or 9000, or 10,000 spots/cm².

In certain instances, the spots on the solid support each represents adifferent capture antibody. In certain other instances, multiple spotson the solid support represent the same capture antibody, e.g., as adilution series comprising a series of descending capture antibodyconcentrations.

Additional examples of methods for preparing and constructing antibodyarrays on solid supports are described in U.S. Pat. Nos. 6,197,599,6,777,239, 6,780,582, 6,897,073, 7,179,638, and 7,192,720; U.S. PatentPublication Nos. 20060115810, 20060263837, 20060292680, and 20070054326;and Varnum et al., Methods Mol. Biol., 264:161-172 (2004).

Methods for scanning antibody arrays are known in the art and include,without limitation, any technique used to scan protein or nucleic acidarrays. Microarray scanners suitable for use in the present inventionare available from PerkinElmer (Boston, Mass.), Agilent Technologies(Palo Alto, Calif.), Applied Precision (Issaquah, Wash.), GSI LumonicsInc. (Billerica, Mass.), and Axon Instruments (Union City, Calif.). As anon-limiting example, a GSI ScanArray3000 for fluorescence detection canbe used with ImaGene software for quantitation.

V. Production of Antibodies

The generation and selection of antibodies not already commerciallyavailable for analyzing the activation state and/or total amount ofsignal transduction molecules in rare circulating cells in accordancewith the present invention can be accomplished several ways. Forexample, one way is to express and/or purify a polypeptide of interest(i.e., antigen) using protein expression and purification methods knownin the art, while another way is to synthesize the polypeptide ofinterest using solid phase peptide synthesis methods known in the art.See, e.g., Guide to Protein Purification, Murray P. Deutcher, ed., Meth.Enzymol., Vol. 182 (1990); Solid Phase Peptide Synthesis, Greg B.Fields, ed., Meth. Enzymol., Vol. 289 (1997); Kiso et al., Chem. Pharm.Bull., 38:1192-99 (1990); Mostafavi et al., Biomed. Pept. ProteinsNucleic Acids, 1:255-60, (1995); and Fujiwara et al., Chem. Pharm.Bull., 44:1326-31 (1996). The purified or synthesized polypeptide canthen be injected, for example, into mice or rabbits, to generatepolyclonal or monoclonal antibodies. One skilled in the art willrecognize that many procedures are available for the production ofantibodies, for example, as described in Antibodies, A LaboratoryManual, Harlow and Lane, Eds., Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1988). One skilled in the art will also appreciatethat binding fragments or Fab fragments which mimic antibodies can alsobe prepared from genetic information by various procedures (see, e.g.,Antibody Engineering: A Practical Approach, Borrebaeck, Ed., OxfordUniversity Press, Oxford (1995); and Huse et al., J. Immunol.,149:3914-3920 (1992)).

In addition, numerous publications have reported the use of phagedisplay technology to produce and screen libraries of polypeptides forbinding to a selected target antigen (see, e.g, Cwirla et al., Proc.Natl. Acad. Sci. USA, 87:6378-6382 (1990); Devlin et al., Science,249:404-406 (1990); Scott et al., Science, 249:386-388 (1990); andLadner et al., U.S. Pat. No. 5,571,698). A basic concept of phagedisplay methods is the establishment of a physical association between apolypeptide encoded by the phage DNA and a target antigen. This physicalassociation is provided by the phage particle, which displays apolypeptide as part of a capsid enclosing the phage genome which encodesthe polypeptide. The establishment of a physical association betweenpolypeptides and their genetic material allows simultaneous massscreening of very large numbers of phage bearing different polypeptides.Phage displaying a polypeptide with affinity to a target antigen bind tothe target antigen and these phage are enriched by affinity screening tothe target antigen. The identity of polypeptides displayed from thesephage can be determined from their respective genomes. Using thesemethods, a polypeptide identified as having a binding affinity for adesired target antigen can then be synthesized in bulk by conventionalmeans (see, e.g., U.S. Pat. No. 6,057,098).

The antibodies that are generated by these methods can then be selectedby first screening for affinity and specificity with the purifiedpolypeptide antigen of interest and, if required, comparing the resultsto the affinity and specificity of the antibodies with other polypeptideantigens that are desired to be excluded from binding. The screeningprocedure can involve immobilization of the purified polypeptideantigens in separate wells of microtiter plates. The solution containinga potential antibody or group of antibodies is then placed into therespective microtiter wells and incubated for about 30 minutes to 2hours. The microtiter wells are then washed and a labeled secondaryantibody (e.g., an anti-mouse antibody conjugated to alkalinephosphatase if the raised antibodies are mouse antibodies) is added tothe wells and incubated for about 30 minutes and then washed. Substrateis added to the wells and a color reaction will appear where antibody tothe immobilized polypeptide antigen is present.

The antibodies so identified can then be further analyzed for affinityand specificity. In the development of immunoassays for a targetprotein, the purified target protein acts as a standard with which tojudge the sensitivity and specificity of the immunoassay using theantibodies that have been selected. Because the binding affinity ofvarious antibodies may differ, e.g., certain antibody combinations mayinterfere with one another sterically, assay performance of an antibodymay be a more important measure than absolute affinity and specificityof that antibody.

Those skilled in the art will recognize that many approaches can betaken in producing antibodies or binding fragments and screening andselecting for affinity and specificity for the various polypeptides ofinterest, but these approaches do not change the scope of the presentinvention.

A. Polyclonal Antibodies

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of a polypeptide ofinterest and an adjuvant. It may be useful to conjugate the polypeptideof interest to a protein carrier that is immunogenic in the species tobe immunized, such as, e.g., keyhole limpet hemocyanin, serum albumin,bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctionalor derivatizing agent. Non-limiting examples of bifunctional orderivatizing agents include maleimidobenzoyl sulfosuccinimide ester(conjugation through cysteine residues), N-hydroxysuccinimide(conjugation through lysine residues), glutaraldehyde, succinicanhydride, SOCl₂, and R₁N═C═NR, wherein R and R₁ are different alkylgroups.

Animals are immunized against the polypeptide of interest or animmunogenic conjugate or derivative thereof by combining, e.g., 100 μg(for rabbits) or 5 μg (for mice) of the antigen or conjugate with 3volumes of Freund's complete adjuvant and injecting the solutionintradermally at multiple sites. One month later, the animals areboosted with about ⅕ to 1/10 the original amount of polypeptide orconjugate in Freund's incomplete adjuvant by subcutaneous injection atmultiple sites. Seven to fourteen days later, the animals are bled andthe serum is assayed for antibody titer. Animals are typically boosteduntil the titer plateaus. Preferably, the animal is boosted with theconjugate of the same polypeptide, but conjugation to a differentimmunogenic protein and/or through a different cross-linking reagent maybe used. Conjugates can also be made in recombinant cell culture asfusion proteins. In certain instances, aggregating agents such as alumcan be used to enhance the immune response.

B. Monoclonal Antibodies

Monoclonal antibodies are generally obtained from a population ofsubstantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possiblenaturally-occurring mutations that may be present in minor amounts.Thus, the modifier “monoclonal” indicates the character of the antibodyas not being a mixture of discrete antibodies. For example, monoclonalantibodies can be made using the hybridoma method described by Kohler etal., Nature, 256:495 (1975) or by any recombinant DNA method known inthe art (see, e.g., U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal (e.g.,hamster) is immunized as described above to elicit lymphocytes thatproduce or are capable of producing antibodies which specifically bindto the polypeptide of interest used for immunization. Alternatively,lymphocytes are immunized in vitro. The immunized lymphocytes are thenfused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form hybridoma cells (see, e.g., Goding,Monoclonal Antibodies: Principles and Practice, Academic Press, pp.59-103 (1986)). The hybridoma cells thus prepared are seeded and grownin a suitable culture medium that preferably contains one or moresubstances which inhibit the growth or survival of the unfused, parentalmyeloma cells. For example, if the parental myeloma cells lack theenzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT), theculture medium for the hybridoma cells will typically includehypoxanthine, aminopterin, and thymidine (HAT medium), which prevent thegrowth of HGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and/or are sensitive to a medium such as HAT medium. Examples ofsuch preferred myeloma cell lines for the production of human monoclonalantibodies include, but are not limited to, murine myeloma lines such asthose derived from MOPC-21 and MPC-11 mouse tumors (available from theSalk Institute Cell Distribution Center; San Diego, Calif.), SP-2 orX63-Ag8-653 cells (available from the American Type Culture Collection;Rockville, Md.), and human myeloma or mouse-human heteromyeloma celllines (see, e.g., Kozbor, J. Immunol., 133:3001 (1984); and Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, pp. 51-63 (1987)).

The culture medium in which hybridoma cells are growing can be assayedfor the production of monoclonal antibodies directed against thepolypeptide of interest. Preferably, the binding specificity ofmonoclonal antibodies produced by hybridoma cells is determined byimmunoprecipitation or by an in vitro binding assay, such as aradioimmunoassay (RIA) or an enzyme-linked immunoabsorbent assay(ELISA). The binding affinity of monoclonal antibodies can be determinedusing, e.g., the Scatchard analysis of Munson et al., Anal. Biochem.,107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(see, e.g., Goding, Monoclonal Antibodies: Principles and Practice,Academic Press, pp. 59-103 (1986)). Suitable culture media for thispurpose include, for example, D-MEM or RPMI-1640 medium. In addition,the hybridoma cells may be grown in vivo as ascites tumors in an animal.The monoclonal antibodies secreted by the subclones can be separatedfrom the culture medium, ascites fluid, or serum by conventionalantibody purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells serveas a preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors, which are then transfected into host cells suchas E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells,or myeloma cells that do not otherwise produce antibody, to induce thesynthesis of monoclonal antibodies in the recombinant host cells. See,e.g., Skerra et al., Curr. Opin. Immunol., 5:256-262 (1993); andPluckthun, Immunol Rev., 130:151-188 (1992). The DNA can also bemodified, for example, by substituting the coding sequence for humanheavy chain and light chain constant domains in place of the homologousmurine sequences (see, e.g., U.S. Pat. No. 4,816,567; and Morrison etal., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide.

In a further embodiment, monoclonal antibodies or antibody fragments canbe isolated from antibody phage libraries generated using the techniquesdescribed in, for example, McCafferty et al., Nature, 348:552-554(1990); Clackson et al., Nature, 352:624-628 (1991); and Marks et al.,J. Mol. Biol., 222:581-597 (1991). The production of high affinity (nMrange) human monoclonal antibodies by chain shuffling is described inMarks et al., BioTechnology, 10:779-783 (1992). The use of combinatorialinfection and in vivo recombination as a strategy for constructing verylarge phage libraries is described in Waterhouse et al., Nuc. AcidsRes., 21:2265-2266 (1993). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma methods forthe generation of monoclonal antibodies.

C. Humanized Antibodies

Methods for humanizing non-human antibodies are known in the art.Preferably, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed by substituting the hypervariable regionsequences of a non-human antibody for the corresponding sequences of ahuman antibody. See, e.g., Jones et al., Nature, 321:522-525 (1986);Riechmann et al., Nature, 332:323-327 (1988); and Verhoeyen et al.,Science, 239:1534-1536 (1988). Accordingly, such “humanized” antibodiesare chimeric antibodies (see, e.g., U.S. Pat. No. 4,816,567), whereinsubstantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome hypervariable region residues and possibly some framework region(FR) residues are substituted by residues from analogous sites of rodentantibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies described herein is an importantconsideration for reducing antigenicity. According to the so-called“best-fit” method, the sequence of the variable domain of a rodentantibody is screened against the entire library of known humanvariable-domain sequences. The human sequence which is closest to thatof the rodent is then accepted as the human FR for the humanizedantibody (see, e.g., Sims et al., J. Immunol., 151:2296 (1993); andChothia et al., J. Mol. Biol., 196:901 (1987)). Another method uses aparticular FR derived from the consensus sequence of all humanantibodies of a particular subgroup of light or heavy chains. The sameFR may be used for several different humanized antibodies (see, e.g.,Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal., J. Immunol., 151:2623 (1993)).

It is also important that antibodies be humanized with retention of highaffinity for the antigen and other favorable biological properties. Toachieve this goal, humanized antibodies can be prepared by a process ofanalysis of the parental sequences and various conceptual humanizedproducts using three-dimensional models of the parental and humanizedsequences. Three-dimensional immunoglobulin models are commonlyavailable and are familiar to those skilled in the art. Computerprograms are available which illustrate and display probablethree-dimensional conformational structures of selected candidateimmunoglobulin sequences. Inspection of these displays permits analysisof the likely role of the residues in the functioning of the candidateimmunoglobulin sequence, i.e., the analysis of residues that influencethe ability of the candidate immunoglobulin to bind its antigen. In thisway, FR residues can be selected and combined from the recipient andimport sequences so that the desired antibody characteristic, such asincreased affinity for the target antigen(s), is achieved. In general,the hypervariable region residues are directly and specifically involvedin influencing antigen binding.

Various forms of humanized antibodies are contemplated in accordancewith the present invention. For example, the humanized antibody can bean antibody fragment, such as a Fab fragment. Alternatively, thehumanized antibody can be an intact antibody, such as an intact IgA,IgG, or IgM antibody.

D. Human Antibodies

As an alternative to humanization, human antibodies can be generated. Insome embodiments, transgenic animals (e.g., mice) can be produced thatare capable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy-chain joining region (JH) gene in chimeric and germ-linemutant mice results in complete inhibition of endogenous antibodyproduction. Transfer of the human germ-line immunoglobulin gene array insuch germ-line mutant mice will result in the production of humanantibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc.Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature,362:255-258 (1993); Bruggermann et al., Year in Immun., 7:33 (1993); andU.S. Pat. Nos. 5,591,669, 5,589,369, and 5,545,807.

Alternatively, phage display technology (see, e.g., McCafferty et al.,Nature, 348:552-553 (1990)) can be used to produce human antibodies andantibody fragments in vitro, using immunoglobulin variable (V) domaingene repertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B cell. Phage display can be performed in avariety of formats as described in, e.g., Johnson et al., Curr. Opin.Struct. Biol., 3:564-571 (1993). Several sources of V-gene segments canbe used for phage display. See, e.g., Clackson et al., Nature,352:624-628 (1991). A repertoire of V genes from unimmunized humandonors can be constructed and antibodies to a diverse array of antigens(including self-antigens) can be isolated essentially following thetechniques described in Marks et al., J. Mol. Biol., 222:581-597 (1991);Griffith et al., EMBO J., 12:725-734 (1993); and U.S. Pat. Nos.5,565,332 and 5,573,905.

In certain instances, human antibodies can be generated by in vitroactivated B cells as described in, e.g., U.S. Pat. Nos. 5,567,610 and5,229,275.

E. Antibody Fragments

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., J. Biochem.Biophys. Meth., 24:107-117 (1992); and Brennan et al., Science, 229:81(1985)). However, these fragments can now be produced directly usingrecombinant host cells. For example, the antibody fragments can beisolated from the antibody phage libraries discussed above.Alternatively, Fab′-SH fragments can be directly recovered from E. colicells and chemically coupled to form F(ab′)₂ fragments (see, e.g.,Carter et al., BioTechnology, 10:163-167 (1992)). According to anotherapproach, F(ab′)₂ fragments can be isolated directly from recombinanthost cell culture. Other techniques for the production of antibodyfragments will be apparent to those skilled in the art. In otherembodiments, the antibody of choice is a single chain Fv fragment(scFv). See, e.g., PCT Publication No. WO 93/16185; and U.S. Pat. Nos.5,571,894 and 5,587,458. The antibody fragment may also be a linearantibody as described, e.g., in U.S. Pat. No. 5,641,870. Such linearantibody fragments may be monospecific or bispecific.

F. Bispecific Antibodies

Bispecific antibodies are antibodies that have binding specificities forat least two different epitopes. Exemplary bispecific antibodies maybind to two different epitopes of the same polypeptide of interest.Other bispecific antibodies may combine a binding site for thepolypeptide of interest with binding site(s) for one or more additionalantigens. Bispecific antibodies can be prepared as full-lengthantibodies or antibody fragments (e.g., F(ab′)₂ bispecific antibodies).

Methods for making bispecific antibodies are known in the art.Traditional production of full-length bispecific antibodies is based onthe co-expression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (see, e.g., Millsteinet al., Nature, 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule is usually performed by affinity chromatography.Similar procedures are disclosed in PCT Publication No. WO 93/08829 andTraunecker et al., EMBO J., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, CH2, and CH3 regions. It is preferred to havethe first heavy chain constant region (CH1) containing the sitenecessary for light chain binding present in at least one of thefusions. DNA encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains into oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm.This asymmetric structure facilitates the separation of the desiredbispecific compound from unwanted immunoglobulin chain combinations, asthe presence of an immunoglobulin light chain in only one half of thebispecific molecule provides for a facile way of separation. See, e.g.,PCT Publication No. WO 94/04690 and Suresh et al., Meth. Enzymol.,121:210 (1986).

According to another approach described in U.S. Pat. No. 5,731,168, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the CH3 domain of an antibody constant domain. In this method,one or more small amino acid side-chains from the interface of the firstantibody molecule are replaced with larger side chains (e.g., tyrosineor tryptophan). Compensatory “cavities” of identical or similar size tothe large side-chain(s) are created on the interface of the secondantibody molecule by replacing large amino acid side-chains with smallerones (e.g., alanine or threonine). This provides a mechanism forincreasing the yield of the heterodimer over other unwanted end-productssuch as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Heteroconjugateantibodies can be made using any convenient cross-linking method.Suitable cross-linking agents and techniques are well-known in the art,and are disclosed in, e.g., U.S. Pat. No. 4,676,980.

Suitable techniques for generating bispecific antibodies from antibodyfragments are also known in the art. For example, bispecific antibodiescan be prepared using chemical linkage. In certain instances, bispecificantibodies can be generated by a procedure in which intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments (see, e.g.,Brennan et al., Science, 229:81 (1985)). These fragments are reduced inthe presence of the dithiol complexing agent sodium arsenite tostabilize vicinal dithiols and prevent intermolecular disulfideformation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody.

In some embodiments, Fab′-SH fragments can be directly recovered from E.coli and chemically coupled to form bispecific antibodies. For example,a fully humanized bispecific antibody F(ab′)₂ molecule can be producedby the methods described in Shalaby et al., J. Exp. Med., 175: 217-225(1992). Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. See, e.g., Kostelny et al., J. Immunol., 148:1547-1553(1992). The leucine zipper peptides from the Fos and Jun proteins werelinked to the Fab′ portions of two different antibodies by gene fusion.The antibody homodimers were reduced at the hinge region to formmonomers and then re-oxidized to form the antibody heterodimers. Thismethod can also be utilized for the production of antibody homodimers.The “diabody” technology described by Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993) has provided an alternativemechanism for making bispecific antibody fragments. The fragmentscomprise a heavy chain variable domain (VH) connected to a light chainvariable domain (VL) by a linker which is too short to allow pairingbetween the two domains on the same chain. Accordingly, the VH and VLdomains of one fragment are forced to pair with the complementary VL andVH domains of another fragment, thereby forming two antigen bindingsites. Another strategy for making bispecific antibody fragments by theuse of single-chain Fv (sFv) dimers is described in Gruber et al., J.Immunol., 152:5368 (1994).

Antibodies with more than two valencies are also contemplated. Forexample, trispecific antibodies can be prepared. See, e.g., Tutt et al.,J. Immunol., 147:60 (1991).

G. Antibody Purification

When using recombinant techniques, antibodies can be produced inside anisolated host cell, in the periplasmic space of a host cell, or directlysecreted from a host cell into the medium. If the antibody is producedintracellularly, the particulate debris is first removed, for example,by centrifugation or ultrafiltration. Carter et al., BioTech.,10:163-167 (1992) describes a procedure for isolating antibodies whichare secreted into the periplasmic space of E. coli. Briefly, cell pasteis thawed in the presence of sodium acetate (pH 3.5), EDTA, andphenylmethylsulfonylfluoride (PMSF) for about 30 min. Cell debris can beremoved by centrifugation. Where the antibody is secreted into themedium, supernatants from such expression systems are generallyconcentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. A protease inhibitor such as PMSF may be included in any of theforegoing steps to inhibit proteolysis and antibiotics may be includedto prevent the growth of adventitious contaminants.

The antibody composition prepared from cells can be purified using, forexample, hydroxylapatite chromatography, gel electrophoresis, dialysis,and affinity chromatography. The suitability of protein A as an affinityligand depends on the species and isotype of any immunoglobulin Fcdomain that is present in the antibody. Protein A can be used to purifyantibodies that are based on human γ1, γ2, or γ4 heavy chains (see,e.g., Lindmark et al., J. Immunol. Meth., 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (see, e.g., Guss etal., EMBO J., 5:1567-1575 (1986)). The matrix to which the affinityligand is attached is most often agarose, but other matrices areavailable. Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a CH3 domain, the Bakerbond ABX™ resin (J. T. Baker;Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, reverse phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™, chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25 M salt).

VI. Examples

The following examples are offered to illustrate, but not to limit, theclaimed invention.

Example 1 Isolation, Stimulation, and Lysis of Circulating Cells

Circulating cells of a solid tumor comprise cells that have eithermetastasized or micrometastasized from a solid tumor and includecirculating tumor cells (CTCs), cancer stem cells (CSCs), and/or cellsthat are migrating to the tumor (e.g., circulating endothelialprogenitor cells (CEPCs), circulating endothelial cells (CECs),circulating pro-angiogenic myeloid cells, circulating dendritic cells,etc.). Patient samples containing circulating cells can be obtained fromany accessible biological fluid (e.g., blood, urine, nipple aspirate,lymph, saliva, fine needle aspirate, etc.). The circulating cells can beisolated from a patient sample using one or more separation methods suchas, for example, immunomagnetic separation (see, e.g., Racila et al.,Proc. Natl. Acad. Sci. USA, 95:4589-4594 (1998); Bilkenroth et al., Int.J. Cancer, 92:577-582 (2001)), microfluidic separation (see, e.g.,Mohamed et al., IEEE Trans. Nanobiosci., 3:251-256 (2004); Lin et al.,Abstract No. 5147, 97th AACR Annual Meeting, Washington, D.C. (2006)),FACS (see, e.g., Mancuso et al., Blood, 97:3658-3661 (2001)), densitygradient centrifugation (see, e.g., Baker et al., Clin. Cancer Res.,13:4865-4871 (2003)), and depletion methods (see, e.g., Meye et al.,Int. J. Oncol., 21:521-530 (2002)).

Manual Isolation of CTCs:

Immunomagnetic Separation of CTCs—Manual Isolation Followed by anActivation Assay:

-   -   1) Magnetic beads (Dynal M450; Dynal AS; Oslo, Norway) that have        been previously conjugated to an anti-EpCAM monoclonal antibody        (Kordia Life Sciences; Leiden, The Netherlands) are used.    -   2) Just prior to use, the pre-coated Dynabeads are washed once        in an equal volume of PBS with BSA at 0.01%.    -   3) 25 μl of the pre-coated Dynabeads are added to 1 ml of the        sample.    -   4) The mixture is incubated for 20 minutes at 2-8° C. with        gentle tilting and rotation.    -   5) The tube is placed in the magnetic separator (MPL-1 magnet)        for 2 minutes.    -   6) The supernatant is discarded and the bead-bound cells are        washed three times by resuspending in PBS with BSA at 0.01%        followed by magnetic separation.    -   7) The sample is resuspended in 100 μl of stimulation buffer.

Sample Preparation:

-   -   1) Peripheral blood from human subjects is drawn in a        siliconized tube containing 1 mg/ml EDTA. The first 3-5 ml is        discarded to avoid contamination with epithelial cells released        from the punctured vein.    -   2) 1 ml of whole blood is diluted 1:3 with 0.9% NaCl prior to        use.

Control Preparation:

-   -   1) Cell line controls are made by spiking human cancer cell        lines into HL-60 cells.    -   2) Cell line controls are used at a concentration of 2.5×10⁶        cells/ml.

Manual Isolation of CECs and CEPCs:

As a non-limiting example, viable CECs and CEPCs can be isolated usingthe immunomagnetic isolation/enrichment technique described in Beerepootet al., Ann. Oncology, 15:139-145 (2004). Briefly, peripheral blood isincubated with magnetic beads (Dynal M450 IgG₁) that have beenpreviously conjugated to an anti-CD146 monoclonal antibody (Kordia LifeSciences). This antibody recognizes all lineages of endothelial cells,but not hematopoetic or epithelial cells, in peripheral blood (George etal., J. Immunol. Meth., 139:65-75 (1991)). Negative selection ofhematopoetic and epithelial cells can be used prior to the positiveselection with magnetic beads conjugated to appropriate antibodies(e.g., Dynal-CD45 beads for depleting leukocytes, Dynal-CD14 beads fordepleting monocytes, Dynal-EpCAM for depleting epithelial cells(Invitrogen; Carlsbad, Calif.)). In this example, only positiveselection is used.

Immunomagnetic Separation of CECs and CEPCs—Manual Isolation Followed byan Activation Assay:

-   -   1) Magnetic beads (Dynal M450) that have been previously        conjugated to an anti-CD146 monoclonal antibody (Kordia Life        Sciences) are used.    -   2) Just prior to use, the pre-coated Dynabeads are washed once        in an equal volume of PBS with BSA at 0.01%.    -   3) 25 μl pre-coated Dynabeads are added to 1 ml of the sample.    -   4) The mixture is incubated for 20 minutes at 2-8° C. with        gentle tilting and rotation.    -   5) The tube is placed in the magnetic separator (MPL-1 magnet)        for 2 minutes.    -   6) The supernatant is discarded and the bead-bound cells are        washed three times by resuspending in PBS with BSA at 0.01%        followed by magnetic separation.    -   7) The sample is resuspended in 100 μl of stimulation buffer.

Sample Preparation:

-   -   1) Peripheral blood from human subjects is drawn in a        siliconized tube containing 1 mg/ml EDTA. The first 3-5 ml is        discarded to avoid contamination with endothelial cells released        from the punctured vein.    -   2) 1 ml of whole blood is diluted 1:3 with 0.9% NaCl prior to        use.

Control Preparation:

-   -   1) Cell line controls are made by spiking human umbilical vein        endothelial cells (HUVEC) into HL-60 cells.    -   2) Cell line controls are used at a concentration of 2.5×10⁶        cells/ml.        Manual Isolation of CEPCs (without CECs):

CEPCs are a circulating subtype of bone marrow-derived progenitor cellsthat have the capacity of differentiating into mature endothelial cellsin response to various angiogenic growth factors. CEPCs may be isolatedby selection with antibodies recognizing the surface marker CD34. CD133is a surface marker that differentiates immature endothelial progenitorcells (EPCs) or primitive hematopoetic stem cells (HSCs) from CEPCs.Various isolation procedures of CEPCs from different sources have beendescribed using adherence culture or magnetic microbeads. In thisexample, a protocol modified from that described in Asahara et al.,Science, 275:964-967 (1997) is used.

Immunomagnetic Separation of CEPCs—Manual Isolation Followed by anActivation Assay:

-   -   1) Magnetic beads (Dynal M450 CD34) are used. These beads are        coated with a monoclonal antibody specific for the CD34 surface        antigen.    -   2) Just prior to use, the pre-coated Dynabeads are washed once        in an equal volume of PBS with BSA at 0.01%.    -   3) 25 μl pre-coated Dynabeads are added to 1 ml of the sample.    -   4) The mixture is incubated for 20 minutes at 2-8° C. with        gentle tilting and rotation.    -   5) The tube is placed in the magnetic separator (MPL-1 magnet)        for 2 minutes.    -   6) The supernatant is discarded and the bead-bound cells are        washed three times by resuspending in PBS with BSA at 0.01%        followed by magnetic separation.    -   7) The sample is resuspended in 100 μl of stimulation buffer.

Sample Preparation:

-   -   1) Peripheral blood from human subjects is drawn in a        siliconized tube containing 1 mg/ml EDTA. The first 3-5 ml is        discarded to avoid contamination with endothelial cells released        from the punctured vein.    -   2) 10 ml of blood is diluted 1:1 with a balanced salt solution.    -   3) 4 ml of diluted blood is layered onto 3 ml of Ficoll-Paque in        10 ml tubes.    -   4) Tubes are spun at 400×g for 30-40 min at 18-20° C.    -   5) The upper layer containing plasma and platelets is drawn off        using a sterile Pasteur pipette, leaving the layer of        mononuclear cells undisturbed at the interface.    -   6) The mononuclear cells are transferred to a sterile centrifuge        tube using a sterile pipette.    -   7) 6 ml of balanced salt solution is added and the cells are        gently resuspended.    -   8) The mixture is centrifuged at 60-100×g for 10 min at 18-20°        C.    -   9) The supernatant is removed and the mononuclear cells from        each tube are resuspended in 1 ml PBS.

Isolation of CTCs, CECs, and CEPCs Using the Veridex System:

Veridex (Warren, N.J.) has commercialized the CellSearch system, whichconsists of a CellPrep system, the CellSearch Epithelial Cell Kit, andthe CellSpotter Analyzer. The CellPrep system is a semi-automated samplepreparation system (Kagan et al., J. Clin. Ligand Assay, 25:104-110(2002)). The CellSearch Epithelial Cell Kit consists of: ferrofluidscoated with anti-EpCAM antibodies specific for epithelial cells;phycoerythrin-conjugated antibodies to cytokeratins 8, 18, and 19; ananti-CD45 antibody conjugated to allophycocyanin; DAPI dye; and buffersfor washing, permeabilizing, and resuspending the cells. The protocolused in this example is also described in Allard et al., Clin. CancerRes., 10:6897-6904 (2004). The entire Veridex system can be used for CTCenumeration or, by removing the sample manually after isolation with theCellPrep system, can provide a method of isolation prior to analysis forpathway activation. The number of CTCs can be informative for algorithmdevelopment.

Veridex System—CTC Enrichment Followed by Enumeration:

-   -   1) 7.5 ml of blood are mixed with 6 ml of buffer, centrifuged at        800×g for 10 minutes, and the placed on the CellPrep system.    -   2) After the instrument aspirates the supernatant, the        instrument adds the ferrofluids.    -   3) The instrument performs the incubation and subsequent        magnetic separation step.    -   4) Unbound cells and the remaining plasma are aspirated.    -   5) Staining reagents are added in conjunction with the        permeabilization buffer for fluorescence staining.    -   6) After incubation by the system, the cells are again separated        magnetically and resuspended in the MagNest Cell Presentation        Device for analysis using the CellSpotter Analyzer.    -   7) The Device is placed on the CellSpotter Analyzer, a        four-color semi-automated fluorescence microscope.    -   8) Images are captured that meet the Veridex defined criteria        and are shown via a web-based browser for final manual        selection.    -   9) Results of cell enumeration are expressed as the number of        cells per 7.5 ml of blood.

Veridex System—CTC Enrichment Followed by an Activation Assay:

-   -   1) 7.5 ml of blood are mixed with 6 ml of buffer, centrifuged at        800×g for 10 minutes, and then placed on the CellPrep system.    -   2) After the instrument aspirates the supernatant, the        instrument adds the ferrofluids.    -   3) The instrument performs the incubation and subsequent        magnetic separation step.    -   4) Unbound cells and the remaining plasma are aspirated.    -   5) The sample is resuspended in 100 μl of stimulation buffer.

Veridex System—CEC and CEPC Enrichment Followed by an Activation Assay:

-   -   1) Veridex offers a CellTracks Endothelial Cell Kit utilizing        capture of CECs and CEPCs with an anti-CD146 antibody. The        CellTracks Endothelial Cell Kit is used in conjunction with        Veridex's CellTracks AutoPrep System for blood sample        preparation and the CellTracks Analyzer II to count and        characterize CECs and CEPCs from whole blood. The protocol is        the same as for the CellSearch Epithelial Cell Kit.

Sample Preparation:

-   -   1) Peripheral blood from human subjects is drawn in the CellSave        Preservative tube according to manufacturer's instructions. The        first 3-5 ml is discarded to avoid contamination with epithelial        or endothelial cells released from the punctured vein.

Manual Isolation of CSCs:

Evidence is building that tumors contain a small population of putativecancer stem cells with unique self-renewal and survival mechanisms (see,e.g., Sells, Crit. Rev. Oncol. Hematol., 51:1-28 (2004); Reya et al.,Nature, 414:105-111 (2001); Dontu et al., Trends Endocrinol. Metal.,15:193-197 (2004); and Dick, Nature, 423:231-233 (2003)). Cancer stemcells (CSCs) may exist in a quiescent state for a long time, making themresistant to chemotherapeutic drugs which target dividing cells. Thiscancer-initiating population can be characterized for activation ofself-renewal and survival pathways subject to targeted therapy forselective removal. Isolation procedures of CSCs have been describedusing adherence culture or magnetic microbeads. In this example, aprotocol modified from that described in Cote et al., Clin. Can. Res.,12:5615 (2006) is used.

Immunomagnetic CSC Isolation—Manual Isolation Followed by an ActivationAssay:

-   -   1) Magnetic beads (Dynal AS; Oslo, Norway) are used. These beads        are coated with a monoclonal antibody specific for either the        CD34 or CD133 surface antigen.    -   2) Just prior to use, the pre-coated Dynabeads are washed once        in an equal volume of PBS with BSA at 0.01%.    -   3) 1-10⁷ pre-coated Dynabeads are added to 3 ml of the sample.    -   4) The mixture is incubated for 60 minutes at 2-8° C. with        gentle tilting and rotating.    -   5) The mixture is divided into 1 ml portions and each tube is        placed in the magnetic separator (MPL-1 magnet) for at least 6        minutes.    -   6) The supernatant is discarded and the bead-bound cells are        washed three times by resuspending in PBS with BSA at 0.01%        followed by magnetic separation.    -   7) The sample is resuspended in 100 μl of stimulation buffer.

Sample Preparation:

-   -   1) Bone marrow specimens are obtained from early breast cancer        patients following patient informed consent.    -   2) Processing the bone marrow aspirates is performed as        described in Bauer et al., Clin. Can. Res., 6:3552-3559 (2000)).        The mononuclear cell fraction containing any disseminated tumor        cells is enriched by Ficoll-Hypaque density gradient        centrifugation using a Beckman GS-6 centrifuge at 4000×g for 35        minutes and washed twice with PBS.

Cell Stimulation and Lysis of Isolated CTCs:

Cell Stimulation:

-   -   1) Growth factors TGF-α (100 nM), heregulin (100 nM), and/or IGF        (100 nM) are added to the cells and incubated at 37° C. for 5        minutes.

Cell Stimulation with Drug Treatment:

-   -   1) Sample is treated with Herceptin, Lapatanib, Tarceva, and/or        Rapamycin analogs at therapeutically effective concentrations        for 30 min. at 37° C.    -   2) Cells are then stimulated by adding growth factors TGF-α (100        nM), heregulin (100 nM), and/or IGF (100 nM) and incubated at        37° C. for 5 minutes.

Cell Stimulation with Drug Treatment (Feedback Loop):

-   -   1) Sample is treated with Herceptin, Lapatanib, Tarceva, and/or        Rapamycin analogs at therapeutically effective concentrations        for 30 min. at 37° C.    -   2) Cells are then stimulated by TGF-α (100 nM), heregulin (100        nM), and/or IGF (100 nM) and incubated at 37° C. for 120        minutes.

Stimulated CTCs are Lysed Using the Following Protocol:

-   -   1) Fresh lysis buffer is freshly prepared by mixing the reagents        set forth in Table 1.    -   2) After the final wash, cells are resuspended on ice in 100 μl        of chilled buffer.    -   3) Incubation is performed on ice for 30 minutes.    -   4) The mixture is spun in a microfuge at maximum speed for 10        minutes to separate the beads from the lysate.    -   5) The lysate is transferred to a new tube for assay or storage        at −80° C.

TABLE 1 Lysis Buffer recipe (10 ml) Reagents Stock conc. Final conc.Volume 10% Triton X-100 10 1 1.00 1 M Tris, pH 7.5 1 0.05 0.05 1 M NaF 10.05 0.05 5 M NaCl 5 0.1 0.20 2 M B-glycerolphosphate 1 0.05 0.50 0.1 MNa₃VO₄ 0.1 0.001 0.10 1 mg/ml pepstatin 1 0.10 Complete mini protease 1tablet 0.5 M EDTA 0.5 0.005 0.10 Total (ml) 3.00 Water (ml) 7.00Cell Stimulation and Lysis of Isolated CECs and/or CEPCs:

VEGF is thought to promote survival by activating antiapoptotic pathwaysin both CEPCs (Larrivee et al., J. Biol. Chem., 278:22006-22013 (2003))and mature CECs, which have been sloughed off the vessel wall (Soloveyet al., Blood, 93:3824-3830 (1999)). VEGF may also stimulate theproliferation of CEPCs or mature CECs, although mature CECs seem to haveonly a limited proliferative capacity compared with CEPCs (Lin et al.,J. Clin. Invest., 105:71-77 (2000)). For these reasons, CECs and/orCEPCs are activated by incubation with VEGF prior to lysis.

Cell Stimulation:

-   -   1) The growth factors VEGF, FGF, PDGF, PIGF, and/or        angiopoietin, each at 100 nM, are added to the cells and        incubated at 37° C. for 5 minutes.

Cell Stimulation with Drug Treatment:

-   -   1) Sample is treated with Avastin, Nexavar, Sutent, and/or        Rapamycin analogs at therapeutically effective concentrations        for 30 min. at 37° C.    -   2) Cells are then stimulated by adding growth factors VEGF, FGF,        PDGF, PIGF, and/or angiopoietin, each at 100 nM, and incubated        at 37° C. for 5 minutes.

Cell Stimulation with Drug Treatment (Feedback Loop):

-   -   1) Sample is treated with Avastin, Nexavar, Sutent, and/or        Rapamycin analogs at therapeutically effective concentrations        for 30 min. at 37° C.    -   2) Cells are then stimulated by adding VEGF, FGF, PDGF, PIGF,        and/or angiopoietin, each at 100 nM, and incubated at 37° C. for        120 minutes.

Isolated CECs and/or CEPC Cells are Lysed Using the Following Protocol:

-   -   1) Fresh lysis buffer is freshly prepared by mixing the reagents        set forth in Table 1.    -   2) After the final wash, cells are resuspended on ice in 100 μl        of chilled buffer.    -   3) Incubation is performed on ice for 30 minutes.    -   4) The mixture is spun in a microfuge at maximum speed for 10        minutes to separate the beads from the lysate.    -   5) The lysate is transferred to a new tube for assay or storage        at −80° C.

Cell Stimulation and Lysis of Isolated CSCs:

Stimulated Cells:

-   -   1) Growth factors TGF-α (100 nM), heregulin (100 nM), and/or IGF        (100 nM) are added to the cells and incubated at 37° C. for 5        minutes.

Stimulated Cells with Drug Treatment:

-   -   1) Sample is treated with Herceptin, Lapatanib, Tarceva, and/or        Rapamycin analogs at therapeutically effective concentrations        for 30 min. at 37° C.    -   2) Cells are then stimulated by adding growth factors TGF-α (100        nM), heregulin (100 nM), and/or IGF (100 nM) and incubated at        37° C. for 5 minutes.

Stimulated Cells with Drug Treatment (Feedback Loop):

-   -   1) Sample is treated with Herceptin, Lapatanib, Tarceva, and/or        Rapamycin analogs at therapeutically effective concentrations        for 30 min. at 37° C.    -   2) Cells are then stimulated by adding growth factors TGF-α (100        nM), heregulin (100 nM), and/or IGF (100 nM) and incubated at        37° C. for 120 minutes.

Isolated CSC Cells are Lysed Using the Following Protocol:

-   -   1) Fresh lysis buffer is freshly prepared by mixing the reagents        set forth in Table 1.    -   2) After the final wash, cells are re-suspended on ice in 100 μl        of chilled buffer.    -   3) Incubation is performed on ice for 30 minutes.    -   4) The mixture is spun in a microfuge at maximum speed for 10        minutes to separate the beads from the lysate.    -   5) The lysate is transferred to a new tube for assay or storage        at −80° C.

Example 2 Single Cell Detection Using a Single Detector Sandwich ELISAwith Tyramide Signal Amplification

This example illustrates a multiplex, high-throughput, single detectorsandwich ELISA having superior dynamic range that is suitable foranalyzing the activation states of signal transduction molecules in rarecirculating cells:

-   -   1) A 96-well microtiter plate was coated with capture antibody        overnight at 4° C.    -   2) The plate was blocked with 2% BSA/TBS-Tween for 1 hour the        next day.    -   3) After washing with TBS-Tween, the cell lysate or recombinant        protein was added at serial dilution and incubated for 2 hours        at room temperature.    -   4) The plate was washed 4 times with TBS-Tween and then        incubated with a biotin-labeled detection antibody for two hours        at room temperature.    -   5) After incubation with the detection antibody, the plate was        washed four times with TBS-Tween and then incubated with        streptavidin-labeled horseradish peroxidase (SA-HRP) for 1 hour        at room temperature to allow the SA-HRP to bind to the        biotin-labeled detection antibody.    -   6) For signal amplification, biotin-tyramide was added at 5        μg/ml with 0.015% H₂O₂ and reacted for 15 minutes.    -   7) After washing six times with TBS-Tween, SA-HRP was added and        incubated for 30 minutes.    -   8) After washing 6 times with TBS-Tween, the HRP substrate TMB        was added and color was developed for 2-10 minutes in the dark.        The reaction was stopped by adding of 0.5M H₂SO₄. The signal was        read on a microplate reader at 450/570 mm.

FIG. 3 shows the detection of total EGFR in A431 cells using an ELISAcomprising monoclonal antibodies against the extracellular domain ofEGFR as the capture antibody and detection antibody. The sensitivity ofthe immunoassay was about 0.25 pg/well based on a recombinantextracellular domain of human EGFR. The calculated EGFR concentrationwas about 0.6 pg in each A431 cell.

FIG. 4 shows the detection of phosphorylated EGFR in A431 cells using anELISA comprising a monoclonal antibody against the extracellular domainof EGFR as the capture antibody and a biotin-labeled monoclonal antibodyagainst phosphorylated EGFR as the detection antibody. Performing a2-fold serial dilution of the capture antibody revealed that there was a1.78-fold increase in signal over background (signal/noise ratio) at theone cell level when the capture antibody concentration was 0.0625 μg/ml.

FIG. 5 shows the detection of total ErbB2 in SKBr3 cells using an ELISAcomprising monoclonal antibodies against the extracellular domain ofErBb2 as the capture antibody and detection antibody. The detectionrange of the immunoassay was between about 1,000 cells and about 1.37cells. There was a 2.71-fold increase in signal over background(signal/noise ratio) at the 1.37 cell level when the capture antibodyconcentration was 1 μg/ml.

FIG. 6 shows the detection of phosphorylated ErBb2 in SKBr3 cells usingan ELISA comprising a monoclonal antibody against the extracellulardomain of ErbB2 as the capture antibody and a monoclonal antibodyagainst phosphorylated ErbB2 as the detection antibody. The detectionrange of the immunoassay was between about 500 cells and about 5 cells.There was a 3.03-fold increase in signal over background (signal/noiseratio) at the 5 cell level when the capture antibody concentration was 1μg/ml.

FIG. 7 shows the detection of total and phosphorylated Erk2 protein inSKBr3 cells using an ELISA comprising monoclonal antibodies against Erk2as the capture antibody and detection antibody. There was a 3.25-foldincrease in signal over background (signal/noise ratio) for total Erk2at the 1.37 cell level. Likewise, there was a 3.17-fold increase insignal over background (signal/noise ratio) for phosphorylated Erk2 atthe 1.37 cell level.

Example 3 Single Cell Detection Using a Single Detector Microarray ELISAwith Tyramide Signal Amplification

This example illustrates a multiplex, high-throughput, single detectormicroarray sandwich ELISA having superior dynamic range that is suitablefor analyzing the activation states of signal transduction molecules inrare circulating cells:

-   -   1) Capture antibody was printed on a 16-pad FAST slide (Whatman        Inc.; Florham Park, N.J.) with a 2-fold serial dilution.    -   2) After drying overnight, the slide was blocked with Whatman        blocking buffer.    -   3) 80 μl of cell lysate was added onto each pad with a 10-fold        serial dilution. The slide was incubated for two hours at room        temperature.    -   4) After six washes with TBS-Tween, 80 μl of biotin-labeled        detection antibody was incubated for two hours at room        temperature.    -   5) After six washes, streptavidin-labeled horseradish peroxidase        (SA-HRP) was added and incubated for 1 hour to allow the SA-HRP        to bind to the biotin-labeled detection antibody.    -   6) For signal amplification, 80 μl of biotin-tyramide at 5 μg/ml        was added and reacted for 15 minutes. The slide was washed six        times with TBS-Tween, twice with 20% DMSO/TBS-Tween, and once        with TBS.    -   7) 80 μl of SA-Alexa 555 was added and incubated for 30 minutes.        The slide was then washed twice, dried for 5 minutes, and        scanned on a microarray scanner (Perkin-Elmer, Inc.; Waltham,        Mass.).

FIG. 8 shows the detection of total EGFR in A431 cells using amicroarray ELISA comprising monoclonal antibodies against theextracellular domain of EGFR as the capture antibody and detectionantibody. A capture antibody dilution curve experiment based upon cellnumbers showed that the microarray ELISA format had a wide dynamic rangeto detect EGFR in about 1-10,000 cells with various concentrations ofcapture antibody in the dilution series. A cell titration curveexperiment based upon the dilution series of capture antibodyconcentrations showed that EGFR could be detected from one cell. Therewas a 2.11-fold increase in signal over background (signal/noise ratio)at the one cell level when the capture antibody concentration was 0.0625mg/ml.

FIG. 9 shows the detection of phosphorylated EGFR in A431 cells using amicroarray ELISA comprising a monoclonal antibody against theextracellular domain of EGFR as the capture antibody and a monoclonalantibody against phosphorylated EGFR as the detection antibody. Acapture antibody dilution curve experiment based upon cell numbersshowed that the microarray ELISA format had a wide dynamic range todetect phosphorylated EGFR in about 1-10,000 cells with variousconcentrations of capture antibody in the dilution series. A celltitration curve experiment based upon the dilution series of captureantibody concentrations showed that phosphorylated EGFR could bedetected from one cell. There was a 1.33-fold increase in signal overbackground (signal/noise ratio) at the one cell level when the captureantibody concentration was 0.125 mg/ml.

FIG. 10 shows the detection of total ErBb2 in SKBr3 cells using amicroarray ELISA comprising monoclonal antibodies against theextracellular domain of ErBb2 as the capture antibody and detectionantibody. A capture antibody dilution curve experiment based upon cellnumbers showed that the microarray ELISA format had a wide dynamic rangeto detect ErBb2 in about 1-10,000 cells with various concentrations ofcapture antibody in the dilution series. A cell titration curveexperiment based upon the dilution series of capture antibodyconcentrations showed that ErBb2 could be detected from one cell. Therewas a 15.27-fold increase in signal over background (signal/noise ratio)at the one cell level when the capture antibody concentration was 0.125mg/ml.

FIG. 11 shows the detection of phosphorylated ErBb2 in SKBr3 cells usinga microarray ELISA comprising a monoclonal antibody against theextracellular domain of ErBb2 as the capture antibody and a monoclonalantibody against phosphorylated ErBb2 as the detection antibody. Acapture antibody dilution curve experiment based upon cell numbersshowed that the microarray ELISA format had a wide dynamic range todetect ErBb2 in about 1-10,000 cells with various concentrations ofcapture antibody in the dilution series. A cell titration curveexperiment based upon the dilution series of capture antibodyconcentrations showed that phosphorylated ErBb2 could be detected fromone cell. There was a 5.45-fold increase in signal over background(signal/noise ratio) at the one cell level when the capture antibodyconcentration was 0.125 mg/ml.

Example 4 Single Cell Detection Using a Proximity Dual DetectorMicroarray ELISA with Tyramide Signal Amplification

This example illustrates a multiplex, high-throughput, proximity dualdetector microarray sandwich ELISA having superior dynamic range that issuitable for analyzing the activation states of signal transductionmolecules in rare circulating cells:

-   -   1) Capture antibody was printed on a 16-pad FAST slide (Whatman        Inc.) with a serial dilution of from 1 mg/ml to 0.004 mg/ml.    -   2) After drying overnight, the slide was blocked with Whatman        blocking buffer.    -   3) 80 μl of A431 cell lysate was added onto each pad with a        10-fold serial dilution. The slide was incubated for two hours        at room temperature.    -   4) After six washes with TBS-Tween, 80 μl of detection        antibodies for the proximity assay diluted in TBS-Tween/2%        BSA/1% FBS was added to the slides. The detection antibodies        used were: (1) an anti-EGFR monoclonal antibody that was        directly conjugated to glucose oxidase (GO); and (2) a        monoclonal antibody recognizing phosphorylated EGFR that was        directly conjugated to horseradish peroxidase (HRP). The        incubation was for 2 hours at room temperature.    -   5) Alternatively, the detection step utilized a biotin-conjugate        of the monoclonal antibody recognizing phosphorylated EGFR. In        these instances, after six washes an additional sequential step        of incubation with streptavidin-HRP for 1 hour was included.    -   6) Alternatively, the detection step utilized an        oligonucleotide-mediated glucose oxidase (GO) conjugate of the        anti-EGFR antibody. Either the directly conjugated or the        biotin-streptavidin (SA) linked conjugate of HRP to the        phosphorylated EGFR antibody was used.    -   6) For signal amplification, 80 μl of biotin-tyramide at 5 μg/ml        was added and reacted for 15 min. The slide was washed six times        with TBS-Tween, twice with 20% DMSO/TBS-Tween, and once with        TBS.    -   7) 80 μl of SA-Alexa 555 was added and incubated for 30 min. The        slide was then washed twice, dried for 5 minutes, and scanned on        a microarray scanner (Perkin-Elmer, Inc.).

FIG. 12 shows a comparison of the proximity dual detector microarrayELISA versus the single detector microarray ELISA. Table 2 shows thesensitivity of the proximity dual detector microarray ELISA versus thesingle detector microarray ELISA. For each A431 cell concentration, thesignal over background (signal/noise ratio) for the proximity and singledetector formats is shown. As illustrated in Table 2, the proximity dualdetector microarray ELISA further increased sensitivity by about 3-foldat the one cell level.

TABLE 2 Single Detector Specific Proximity Format Format Cell No. SignalSignal (S/N Ratio) (S/N Ratio) 100 547 465 6.6 2.1 10 388 306 4.7 1.3 1295 213 3.6 1.3 0 82

FIG. 13 shows the assay specificity for the single detector microarrayELISA versus the proximity dual detector microarray ELISA. Experimentswhich generated titration curves of phosphorylated EGFR at variouscapture antibody concentrations in the single detector format exhibitedvery high background due to the lack of specificity of the singledetection antibody. In contrast, experiments which generated titrationcurves of phosphorylated EGFR at various capture antibody concentrationsin the proximity dual detector format exhibited very low background dueto the increased specificity obtained by detecting the proximity betweentwo detection antibodies.

Example 5 Single Cell Detection of the Activation States of a Pluralityof Signal Transducers Using Addressable Proximity Dual DetectorMicroarrays

This example illustrates a multiplex, high-throughput, addressableproximity dual detector microarray assay having superior dynamic rangethat is suitable for analyzing the activation states of a plurality ofsignal transduction molecules in rare circulating cells:

-   -   1) Capture antibodies were printed on a 16-pad FAST slide        (Whatman Inc.). The capture antibodies printed were EGFR, HER2,        Erk, Shc, PI3K, and pan-cytokeratin. A 2-fold dilution series of        each capture antibody (0.25 mg/ml, 0.125 mg/ml, and 0.0625        mg/ml) was used, and double and quadruple spots were made for        each antibody dilution.    -   2) After drying overnight, the slide was blocked with Whatman        blocking buffer.    -   3) 80 μl of cell lysate was added onto each pad with a 10-fold        serial dilution. The slide was incubated for two hours at room        temperature.    -   4) After six washes with TBS-Tween, 80 μl of detection        antibodies for the proximity assay diluted in TBS-Tween/2%        BSA/1% FBS was added to the slides. The detection antibodies        used were: (1) an anti-EGFR monoclonal antibody that was        directly conjugated to glucose oxidase (GO); and (2) a        monoclonal antibody recognizing phosphorylated EGFR that was        directly conjugated to HRP. The incubation was for 2 hours at        room temperature.    -   5) Alternatively, the detection step utilized a biotin-conjugate        of the monoclonal antibody recognizing phosphorylated EGFR. In        these instances, after six washes an additional sequential step        of incubation with streptavidin-HRP for 1 hour was included.    -   6) Alternatively, the detection step utilized an        oligonucleotide-mediated glucose oxidase conjugate of the        anti-EGFR antibody. Either the directly conjugated or        biotin-streptavidin (SA) linked conjugate of HRP to the        phosphorylated EGFR antibody was used.    -   7) To detect total HER2 and phosphorylated protein, steps 4),        5), or 6) were performed using a monoclonal antibody recognizing        HER2 in place of the monoclonal antibody recognizing EGFR.    -   8) For signal amplification, 80 μl of biotin-tyramide at 5 μg/ml        was added and reacted for 15 min. The slide was washed six times        with TBS-Tween, twice with 20% DMSO/TBS-Tween, and once with        TBS.    -   9) 80 μl of SA-Alexa 555 was added and incubated for 30 min. The        slide was then washed twice, dried for 5 minutes, and scanned on        a microarray scanner (Perkin-Elmer, Inc.).

FIG. 14 shows an exemplary embodiment of the format of the addressablemicroarray. Five targets are addressable via specific capture antibodies(e.g., EGFR, HER2, Shc, Erk, and PI3K). Phosphorylated complexes of Shc,Erk, or PI3K with either EGFR or HER2 can be detected on this arrayusing the proximity dual detector format. Pan-cytokeratin (PanCK) servesas a control to normalize for the number of epithelial cells.

FIG. 15 shows the detection of phosphorylated Shc levels in a titrationanalysis of stimulated A431 cells. The addressable array simultaneouslyprovided information on EGFR and HER2 phosphorylation.

Example 6 Dynamic Range Extension of Proximity Dual Detector Microarrays

This example illustrates that the dynamic range for analyzing theactivation states of signal transduction molecules in rare circulatingcells can be enhanced by performing a dilution series on the captureantibody in a multiplex, high-throughput, proximity dual detectormicroarray assay:

-   -   1) Capture antibody was printed on a 16-pad FAST slide (Whatman        Inc.). Each capture antibody was serially diluted 2-fold for a        total of nine concentrations (1 mg/ml starting; 0.004 mg/ml        ending).    -   2) After drying overnight, the slide was blocked with Whatman        blocking buffer.    -   3) 80 μl of cell lysate was added onto each pad with a 10-fold        serial dilution. The slide was incubated for two hours at room        temperature.    -   4) After six washes with TBS-Tween, 80 μl of detection        antibodies for the proximity assay diluted in TBS-Tween/2%        BSA/1% FBS was added to the slides. The detection antibodies        used were: (1) an anti-EGFR monoclonal antibody that was        directly conjugated to glucose oxidase (GO); and (2) a        monoclonal antibody recognizing phosphorylated EGFR that was        directly conjugated to HRP. The incubation was for 2 hours at        room temperature.    -   5) Alternatively, the detection step utilized a biotin-conjugate        of the monoclonal antibody recognizing phosphorylated EGFR. In        these instances, after six washes an additional sequential step        of incubation with streptavidin-HRP for 1 hour was included.    -   6) Alternatively, the detection step utilized an        oligonucleotide-mediated glucose oxidase conjugate of the        anti-EGFR antibody. Either the directly conjugated or        biotin-streptavidin (SA) linked conjugate of HRP to the        phosphorylated EGFR antibody was used.    -   7) For signal amplification, 80 μl of biotin-tyramide at 5 μg/ml        was added and reacted for 15 min. The slide was washed six times        with TBS-Tween, twice with 20% DMSO/TBS-Tween, and once with        TBS.    -   8) 80 μl of SA-Alexa 555 was added and incubated for 30 min. The        slide was then washed twice, dried for 5 minutes, and scanned on        a microarray scanner (Perkin-Elmer, Inc.).

FIG. 16 shows the dilution curves of an anti-EGFR capture antibody.Using addressable arrays, detection of phosphorylated and total EGFR wasat the single cell level for stimulated A431 cells. The dynamic range ofthis assay was greater than 5 logs. Each individual curve had a dynamicrange of about 2 logs, but the dynamic range was significantly enhancedwhen the information from the 6 informative curves was combined.

Example 7 Oligonucleotide Conjugation to Antibodies

This example illustrates a conjugation and quality control procedure forgenerating oligonucleotide-conjugated antibodies or enzymes.

Conjugation:

-   -   1) 72-mer oligonucleotide linkers were synthesized with a 5′-SH        group and a 6-carbon spacer.    -   2) The lyophilized oligonucleotide linkers were dissolved in 20        mM Tris-HCl, pH 7.4. 125 mmoles of linkers were then treated        with 0.5M TCEP-HCl (Pierce; Rockford, Ill.) at a final        concentration of 50 mM at room temperature for 2 hrs. Tris,        TCEP, and unconjugated linkers in the reaction mixture were        eliminated using a desalting spin column.    -   3) The resulting deprotected oligonucleotide linkers were        conjugated to the primary amines of target proteins (e.g.,        antibodies or enzymes) using a heterobifunctional cross-linker        such as SMCC in a 100 μl reaction volume.    -   4) The reaction mix was incubated at room temperature for 2 hrs.        The oligonucleotide-conjugated antibodies or enzymes were        purified using gel filtration with a Sephacryl S-200 HR column        (GE Healthcare; Piscataway, N.J.).

Conjugate Qualification:

-   -   1) After glucose oxidase (GO) molecules were conjugated to a        first oligonucleotide linker, three fractions of the resulting        GO-oligonucleotides were collected after purification and were        printed on nitrocellulose-coated slides in a 10-fold dilution        series.    -   2) IgG was conjugated to Alexa 647 and a second oligonucleotide        linker having a sequence complementary to the first        oligonucleotide linker. The resulting Alexa        647-oligonucleotide-conjugated antibodies were applied onto the        chip and hybridized in 1×PBS buffer for 1 hr at room temperature        and washed several times.    -   3) The slides were dried and scanned with a microarray scanner        to confirm nucleotide sequence-specific hybridization.    -   4) A glucose oxidase enzymatic assay confirmed that the        conjugation process did not alter the function of the enzyme.

FIG. 17 shows that the Alexa 647-oligonucleotide-conjugated antibodieshad the highest binding affinity for the GO-oligonucleotides infractions 13-15.

Example 8 Oligonucleotide-Conjugated Antibodies for SimultaneousDetection of Total and Phosphorylated EGFR

This example illustrates a multiplex, high-throughput, microarray assayfor analyzing the activation states of signal transduction molecules inrare circulating cells using the oligonucleotide conjugates described inExample 7:

-   -   1) Capture antibody was printed on a 16-pad FAST slide (Whatman        Inc.).    -   2) After drying overnight, the slide was blocked with Whatman        blocking buffer.    -   3) 80 μl of cell lysate was added onto each pad with a 10-fold        serial dilution. The slide was incubated for two hours at room        temperature.    -   4) After six washes with TBS-Tween, 80 μl of detection        antibodies for the proximity assay diluted in TBS-Tween/2%        BSA/1% FBS was added to the slides. The detection antibodies        used were: (1) an anti-EGFR monoclonal antibody that was        directly conjugated to Alexa 647 and an oligonucleotide linker,        wherein the oligonucleotide linker comprises a sequence        complementary to an oligonucleotide linker conjugated to glucose        oxidase (GO); and (2) a monoclonal antibody recognizing        phosphorylated EGFR that was directly conjugated to horseradish        peroxidase (HRP). The Alexa 647-oligonucleotide-conjugated        anti-EGFR antibody was contacted with the GO-oligonucleotide to        form the complex shown in FIG. 18. Excess unbound reagents were        removed by washing six times with TBS-Tween.    -   5) Glucose was then added to the reaction along with        tyramide-Alexa 555.    -   6) Total EGFR levels were detected by direct binding of the        Alexa 647-oligonucleotide-conjugated anti-EGFR antibody.        Phosphorylated EGFR was detected by the proximity binding of the        GO-oligonucleotide to the HRP-conjugated anti-p-EGFR antibody        and visualized by tyramide signal amplification.    -   7) The slide was scanned with a laser specific for Alexa 647 and        Alexa 555 on a microarray scanner (Perkin-Elmer, Inc.).

FIG. 19 shows the simultaneous detection of total EGFR andphosphorylated EGFR. Total EGFR (t-EGFR) was detected by a directbinding assay from as few as 10 cells and phosphorylated EGFR (p-EGFR)was detected from 1 cell. 10e⁵ p-EGFR molecules were detected with theproximity signal amplification method. The detection limit of p-EGFR wasincreased over 100-fold by using the proximity assay format.

Example 9 Detection of Circulating Tumor Cell (CTC) Signaling in BreastCancer Patients

Microarray fabrication and processing are adapted from methods describedby Chan et al., Nat. Med., 10:1390-1396 (2004). Antibodies (1 mg/ml)against the signal transducers EGFR, ErbB2, ErbB3, ErbB4, IGF-1R, Akt,Erk, p70S6K, Bad, Rsk, Mek, cSrc, cytokeratin, tubulin, β-actin, andanti-mouse antibody (Positive Control) (4/4 array) are transferred to a384-well polypropylene plate (50 μl/well) using a contact printingrobotic microarrayer (Bio-Rad Laboratories; Hercules, Calif.) fittedwith solid spotting pins to spot antibodies onto FAST® Slides (WhatmanInc.; Florham Park, N.J.). Slides coated with 8 sectored pads are used.After printing, the slides are blocked with a 3% casein solution. Slidesare stored at least overnight under dry conditions before use.

Patient selection criteria, sample preparation methods, and study designare adapted from published studies evaluating circulating tumor cells inwomen with suspected breast cancer (see, e.g., Wulfing et al., Clin.Cancer Res., 12:1715-1720 (2006); and Reinholz et al., Clin. CancerRes., 11:3722-3732 (2005)). Women with a breast abnormality detected onimaging and who are to undergo a breast biopsy are approached for thisstudy. At least forty-two patients who are diagnosed with primary breastcancer are included. At least thirty-five patients have no sign of overtmetastasis at the time of primary diagnosis. At least seven patientshave distant metastases at diagnosis and are considered a positivereference group. None of the patients have a history of previous cancer.Age and treatment information (e.g., surgical therapy, chemotherapy,radiotherapy, endocrine therapy, etc.) is collected for each patient.Approximately 20 ml of blood is collected from each patient and allblood samples are assigned a unique identification number. All assaysare done with the investigators blinded to the results of the biopsy.

Peripheral blood (18 ml) is added to an Accuspin Histopaque-1077 system(Sigma Aldrich; St. Louis, Mo.) and centrifuged at 1,500 rpm for 10minutes in a Beckman CS-6R tabletop centrifuge (Beckman Instruments;Palo Alto, Calif.). The mononuclear cell layer is removed, washed twicewith PBS, and diluted to 1 ml with PBS/0.1% bovine serum albumin. Theepithelial cells are enriched by immunomagnetic capture using antibodiesagainst Ber-EP4 attached to magnetic beads using the DynabeadsEpithelial Enrich kit according to the manufacturer's instructions(Dynal AS; Oslo, Norway). The cells are mixed with 1-107 beads in avolume of 20 ml while rocking for 1 hour. The Ber-EP4 antibodyrecognizes two glycoproteins on the surface and in the cytoplasm ofepithelial cells, except the superficial layers of squamous epithelialcells, hepatocytes, and parietal cells. The suspension is placed on amagnet for at least 6 minutes and the supernatant is carefully removed.The cells attached to the magnetic beads are washed thrice with 1 mlPBS/0.1% bovine serum albumin. Growth factors TGF-α (100 nM), heregulin(100 nM), and IGF (100 nM) are added to the cells and incubated at 37°C. for 5 minutes. The cells are concentrated and lysed with the lysisbinding buffer supplied with the kit. The lysed cell suspension (withbeads attached) is stored at 80° C. until processing.

Assay Method 1: Lysates (40 μl) are applied to the array, incubatedovernight, and washed three times with wash buffer. HRP-labeledanti-phospho antibody (conjugated as described, e.g., in Kuhlmann,Immuno Enzyme Techniques, Verlag Chemie, Weinheim, pp 1-162 (1984))against each of the signal transducers and anti-total antibody (i.e.,activation state-independent) labeled with glucose oxidase (conjugatedas described in Kuhlmann, supra) are added to the array, incubated for 2hours, and washed three times with wash buffer. Tyramide reagent(Molecular Probes) and glucose are added and the reaction is developedfor one hour and washed three times. The array is incubated withstreptavidin-HRP for 30 minutes and washed and developed using enhancedluminol (Molecular Probes). Signal is detected using a CCD camera.

Assay Method 2: Antibodies (1 mg/ml) against 2,4-dinitrophenol (DNP) aretransferred to a 384-well polypropylene plate (5 μl/well) using acontact printing robotic microarrayer (Bio-Rad Laboratories; Hercules,Calif.) fitted with solid spotting pins to spot antibodies onto FAST®Slides (Whatman Inc.; Florham Park, N.J.). Slides coated with 8 sectoredpads are used. After printing, the slides are blocked with a 3% caseinsolution. Slides are stored at least overnight under dry conditionsbefore use.

The lysate (40 μl) is mixed with total DNP-labeled antibody against eachof the above signal transducers, anti-phospho antibody labeled withOligo and Alexa Fluor® 647, and anti-total antibody labeled with Oligoand Alexa Fluor® 647, added to the anti-DNP antibodies, incubatedovernight, and washed three times with wash buffer. 2,4-dinitro lysine(Molecular Probes) is added to release the immune complexes from theanti-DNP antibodies. The released immune complexes are added to azip-code array (see, e.g., Keramas et al., Lab Chip, 4:152-158 (2004);and Delrio-Lafreniere et al., Diagn. Microbiol. Infect. Dis., 48:23-31(2004)) and incubated overnight. The array is washed three times and theprocessed slides are scanned using a GenePix 4000A microarray scanner(Axon Scanner) at 10 micron resolution.

Example 10 Detection of Circulating Endothelial Cell (CEC) andCirculating Endothelial Precursor Cell (CEP) Signaling in Breast CancerPatients

The same patient samples, sample preparation, and assay methodsdescribed in Example 9 are used, except that CEC and CEP cells areenriched by immunomagnetic capture using the monoclonal antibody P1H12or CD146, attached to magnetic Dynabeads. A microarray is fabricated andprocessed using the methods described in Example 9, except that thearrayed antibodies are specific for the following analytes: VEGFR1,VEGFR2, VEGFR3, TIE1, TIE2, PDGFR-α, PDGFR-β, FGFR1, FGFR2, Akt, Erk,p70S6K, Rsk, cSrc, and β-actin. The assay methods described in Example 9are used to detect CEC and CEP signaling.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. Although the foregoing invention has beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, it will be readily apparent to those ofordinary skill in the art in light of the teachings of this inventionthat certain changes and modifications may be made thereto withoutdeparting from the spirit or scope of the appended claims.

1. A method for performing a multiplex, high-throughput immunoassayhaving superior dynamic range, the method comprising: (a) incubating acellular extract with a plurality of dilution series of captureantibodies specific for one or more analytes in the cellular extract toform a plurality of captured analytes, wherein the capture antibodiesare restrained on a solid support; (b) incubating the plurality ofcaptured analytes with detection antibodies specific for thecorresponding analytes to form a plurality of detectable capturedanalytes, wherein the detection antibodies comprise: (1) a plurality ofactivation state-independent antibodies labeled with a facilitatingmoiety, and (2) a plurality of activation state-dependent antibodieslabeled with a first member of a signal amplification pair, wherein thefacilitating moiety generates an oxidizing agent which channels to andreacts with the first member of the signal amplification pair; (c)incubating the plurality of detectable captured analytes with a secondmember of the signal amplification pair to generate an amplified signal;and (d) detecting the amplified signal generated from the first andsecond members of the signal amplification pair.
 2. The method of claim1, wherein the cellular extract comprises an extract of circulatingcells of a solid tumor.
 3. The method of claim 2, wherein the cells areisolated from a patient sample by immunomagnetic separation.
 4. Themethod of claim 3, wherein the patient sample is selected from the groupconsisting of whole blood, serum, plasma, urine, sputum, bronchiallavage fluid, tears, nipple aspirate, lymph, saliva, fine needleaspirate, and combinations thereof.
 5. The method of claim 3, whereinthe isolated cells are selected from the group consisting of circulatingtumor cells, circulating endothelial cells, circulating endothelialprogenitor cells, cancer stem cells, and combinations thereof.
 6. Themethod of claim 3, wherein the isolated cells are stimulated in vitrowith growth factors.
 7. The method of claim 6, wherein the isolatedcells are incubated with an anticancer drug prior to growth factorstimulation.
 8. The method of claim 7, wherein the anticancer drug isselected from the group consisting of a monoclonal antibody, tyrosinekinase inhibitor, immunosuppressive agent, and combinations thereof. 9.The method of claim 6, wherein the isolated cells are lysed followinggrowth factor stimulation to produce the cellular extract.
 10. Themethod of claim 1, wherein the one or more analytes comprise a pluralityof signal transduction molecules.
 11. The method of claim 1, wherein thesolid support is selected from the group consisting of glass, plastic,chips, pins, filters, beads, paper, membrane, fiber bundles, andcombinations thereof.
 12. The method of claim 1, wherein the activationstate-independent antibodies further comprise a detectable moiety. 13.The method of claim 12, wherein the detectable moiety is a fluorophore.14. The method of claim 12, wherein the amount of the detectable moietyis correlative to the amount of one or more of the analytes.
 15. Themethod of claim 1, wherein the activation state-independent antibodiesare directly labeled with the channeling moiety.
 16. The method of claim1, wherein the activation state-independent antibodies are labeled withthe channeling moiety via hybridization between an oligonucleotideconjugated to the activation state-independent antibodies and acomplementary oligonucleotide conjugated to the channeling moiety. 17.The method of claim 1, wherein the activation state-dependent antibodiesare directly labeled with the first member of the signal amplificationpair.
 18. The method of claim 1, wherein the activation state-dependentantibodies are labeled with the first member of the signal amplificationpair via binding between a first member of a binding pair conjugated tothe activation state-dependent antibodies and a second member of thebinding pair conjugated to the first member of the signal amplificationpair.
 19. The method of claim 18, wherein the first member of thebinding pair is biotin.
 20. The method of claim 18, wherein the secondmember of the binding pair is streptavidin.
 21. The method of claim 1,wherein the channeling moiety is glucose oxidase.
 22. The method ofclaim 21, wherein the oxidizing agent is hydrogen peroxide (H₂O₂). 23.The method of claim 22, wherein the first member of the signalamplification pair is a peroxidase.
 24. The method of claim 23, whereinthe peroxidase is horseradish peroxidase (HRP).
 25. The method of claim23, wherein the second member of the signal amplification pair is atyramide reagent.
 26. The method of claim 25, wherein the tyramidereagent is biotin-tyramide.
 27. The method of claim 26, wherein theamplified signal is generated by peroxidase oxidization of thebiotin-tyramide to produce an activated tyramide.
 28. The method ofclaim 27, wherein the activated tyramide is directly detected.
 29. Themethod of claim 27, wherein the activated tyramide is detected upon theaddition of a signal-detecting reagent.
 30. The method of claim 29,wherein the signal-detecting reagent is a streptavidin-labeledfluorophore.
 31. The method of claim 29, wherein the signal-detectingreagent is a combination of a streptavidin-labeled peroxidase and achromogenic reagent.
 32. The method of claim 31, wherein the chromogenicreagent is 3,3′,5,5′-tetramethylbenzidine (TMB).
 33. An array havingsuperior dynamic range comprising a plurality of dilution series ofcapture antibodies specific for one or more analytes in a cellularextract, wherein the capture antibodies are restrained on a solidsupport. 34.-47. (canceled)
 35. A method for performing a multiplex,high-throughput immunoassay having superior dynamic range, the methodcomprising: (a) incubating a cellular extract with a plurality ofdilution series of capture antibodies specific for one or more analytesin the cellular extract to form a plurality of captured analytes,wherein the capture antibodies are restrained on a solid support; (b)incubating the plurality of captured analytes with detection antibodiesspecific for the corresponding analytes to form a plurality ofdetectable captured analytes; (c) incubating the plurality of detectablecaptured analytes with first and second members of a signalamplification pair to generate an amplified signal; and (d) detecting anamplified signal generated from the first and second members of thesignal amplification pair. 36.-72. (canceled)